CN109929818A - Modified phenylalanine oxidizing ferment proenzyme and application thereof - Google Patents

Modified phenylalanine oxidizing ferment proenzyme and application thereof Download PDF

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CN109929818A
CN109929818A CN201910265832.9A CN201910265832A CN109929818A CN 109929818 A CN109929818 A CN 109929818A CN 201910265832 A CN201910265832 A CN 201910265832A CN 109929818 A CN109929818 A CN 109929818A
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gly
leu
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CN109929818B (en
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王辰
肖飞
张俊华
许小毛
邹丽辉
李传保
王萌
苏斐
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Beijing Hospital
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Abstract

The application provides a kind of modified phenylalanine oxidizing ferment proenzyme and its purposes for being used for proteinase activity detection.It is used on the way at this, each testing protein enzyme concentration signal is effectively amplified, and the sensitivity of detection is improved, and realizes special, sensitive, the quick detection of vivo protein enzymatic activity.

Description

Modified phenylalanine oxidizing ferment proenzyme and application thereof
Technical field
This application involves a kind of modified proenzymes and application thereof, belong to proteinase activity detection field.
Background technique
There are multiple protein enzymes in human body, however lack and a kind of can be widely applied for detecting the straight of various proteinase activities It connects, is special, is sensitive, is quick, effective method.
By taking blood coagulation system as an example, the factor IIa in system is also referred to as fibrin ferment, belongs to serine protease, is blood The key link of thrombus and hemostasis in procoagulant system.Fibrin ferment in blood circulation exists in the form of inactive factor, The only active fibrin ferment containing denier in normal human.Currently, the detection method of fibrin ferment is roughly divided into indirect detection Method includes measurement Prothrombin fragment1+2 (F1+2), fibrinopeptide A, soluble fibrin monomer complex, fibrin ferment- Antithrombin compounds, thromboplastin generation etc.；Direct Detection Method includes Chromogenic assay and fluorescence method.However, due to activation Only have more than ten to tens number difference, above-mentioned method between state and the coagulation factor of unactivated state on amino acid sequence It is difficult to distinguish it.In addition, the indirect detection method of fibrin ferment is using relatively broad at present, but its sensibility and specificity It is relatively poor.It is active related to yet there are no physiological status and the prothrombin a of state of activation in directly detection blood Report.
Intracorporal fibrinolytic system and blood coagulation system are closely connected, plasminogen (Plasminogen) quilt in fibrinolytic system After streptokinase or urokinase activated become fibrinolysin (Plasmin), the latter can single-minded fibrin degradation gel, so that it is decomposed At soluble product, the level of internal fibrinogen is reduced by negative-feedback effect, to avoid fibrinous excessive solidifying It is poly-.It yet there are no the relevant report of the plasmin activity of physiological status and state of activation in directly detection blood.
The testing result reaction of coagulogram be blood coagulation system a kind of overall performance, be difficult to navigate to some blood coagulation because The situation of son.And the ELISA method about existing detection coagulation factor, only detecting specific protein whether there is and its contain Amount, and there is no the activity of reaction enzymes.In addition, the antibody titer and specificity issues in this method limit the mark of testing result Standardization.
It needs exist for a kind of can be widely applied for the direct, sensitive, quick, special of various proteinase activities detection in human body Different, effective method.
Summary of the invention
On the one hand, the present invention provides a kind of modified phenylalanine oxidizing ferment proenzyme (the proenzyme of L- Phenylalanine oxidase, is abbreviated as proPAO), it is characterised in that: the modification is in phenylalanine oxidizing ferment proenzyme Introduce between leader sequence in sequence and α subunit and between α subunit and β subunit the specific recognition site sequence for having protease.
In some embodiments, phenylalanine oxidizing ferment proenzyme derives from bacterium, such as Pseudomonas Sp.P- 501, East Lake pseudomonad (Pseudomonas donghuensis), Ralstonia solanacearum (Ralstonia Solanacearum) UW551 or onion Burkholderia (Burkholderia cepacia) ATCC 25416.
In some embodiments, protease be selected from coagulation cascade protease, such as prothrombin a, Va, VIIa, VIIIa, IXa, Xa, XIa or XIIa；Fibrinolysin；Aspartic protease containing cysteine, such as Caspase-1, Caspase-2、Caspase-3、Caspase-4、Caspase-5、Caspase-6、Caspase-7、Caspase-8、 Caspase-9, Caspase-10 or Caspase-11；Complement pathway protein enzyme, for example, factor C1, C2, C3, C4, C5, C6, C7, C8, C9 or C3/C5 converting Enzyme；MMP family protein enzyme；NisP enzyme.
Technical staff knows how to determine the specific recognition site sequence of protease, and knows albumen known in the art The specific recognition site sequence of enzyme.
In some embodiments, the specific recognition sites sequence of the protease such as SEQ ID NO:8-44,67 any It is shown, for example, SEQ ID NO:9-12 it is any shown in.
For example, the cutting sequence of prothrombin a include but is not limited to Xaa Xaa Xab Arg Xac (SEQ ID NO: 8), cleavage site is between Arg and Xac, wherein " Xaa " is arbitrary amino acid residue, " Xab " is preferably to be selected from Pro, Ala, Gly Or the amino acid residue of Val, " Xac " are the amino acid residue for preferably being selected from Ser, Ala, Gly, preferably Leu Val Pro Arg Gly(SEQ ID NO:10)；The cutting sequence of prothrombin a may be Leu Arg Pro Arg (SEQ ID NO:9) (cleavage site is after Arg), Phe Pro Arg (SEQ ID NO:11) (cleavage site is after Arg) and Gly Arg Gly (SEQ ID NO:12) (cleavage site is between Arg and Gly) etc..The cutting sequence of proconvertin a includes but is not limited to Leu Ile Gln Arg(SEQ ID NO:13).The cutting sequence of factor IXa includes but is not limited to Xaa Xaa Gly Arg (SEQ ID NO:14), " Xaa " are arbitrary amino acid residue, preferably Pro Gln Gly Arg (SEQ ID NO:15).It is solidifying The cutting sequence of blood factor Xa includes but is not limited to Xad Xaa Xae Xaf (SEQ ID NO:16), wherein " Xaa " is any ammonia Base acid residue, " Xad " are the amino acid residue for preferably being selected from Ala or Ile, and " Xae " is the amino acid for preferably being selected from Pro, Phe or Gly Residue, " Xaf " are the amino acid residue selected from Arg or Lys, preferably Ile Glu Gly Arg (SEQ ID NO:17) and Ile Asp Gly Arg (SEQ ID NO:18) etc..The cutting sequence of plasma thromboplastin antecedent a includes but is not limited to Xag Xah Thr Arg (SEQ ID NO:19), wherein " Xag " is the amino acid residue for preferably being selected from Lys or Asp, " Xah " is preferably to be selected from Phe's or Leu Amino acid residue, preferably Lys Leu Thr Arg (SEQ ID NO:20).The cutting sequence of Hageman factor a includes but unlimited In Thr Ser Thr Arg (SEQ ID NO:21).The cutting sequence of fibrinolysin Plasmin includes but is not limited to Xaa Xaa Xai Xaj (SEQ ID NO:22), cleavage site is between Xai and Xaj, wherein " Xaa " is arbitrary amino acid residue, " Xai " For the amino acid residue for preferably being selected from Arg or Lys, " Xaj " is the amino acid residue for preferably being selected from Ala, Ser, Gly or Arg, preferably Gly Tyr Arg Ala (SEQ ID NO:23) and Pro Ala Lys Ala (SEQ ID NO:24).The cutting of Caspase-2 Sequence includes but is not limited to Asp Glu Xaa Asp (SEQ ID NO:25), wherein " Xaa " is arbitrary amino acid residue. The cutting sequence of Caspase-3 includes but is not limited to Xak Glu Val Asp (SEQ ID NO:26), wherein " Xak " is preferred From the amino acid residue of Asp or Glu, preferably Asp Glu Val Asp (SEQ ID NO:27).The cutting sequence of Caspase-6 Including but not limited to Xaa Glu Xaa Asp (SEQ ID NO:28), wherein " Xaa " is arbitrary amino acid residue.Caspase-7 Cutting sequence include but is not limited to Asp Xaa Xaa Asp (SEQ ID NO:29), wherein " Xaa " be arbitrary amino acid it is residual Base.The cutting sequence of Caspase-8 includes but is not limited to Xal Xam Thr Asp (SEQ ID NO:30), wherein " Xal " is excellent Amino acid residue selected from Asp or Leu, " Xam " are the amino acid residue for preferably being selected from Glu or Ser, preferably Asp Glu Thr Asp(SEQ ID NO:31).The cutting sequence of Caspase-9 include but is not limited to Xaa Xan Xaa Asp (SEQ ID NO: 32), wherein " Xaa " is arbitrary amino acid residue, and " Xan " is the amino acid residue for preferably being selected from Asp or Glu.Caspase-10's Cutting sequence includes but is not limited to Xaa Xao Xap Asp (SEQ ID NO:33), wherein and " Xaa " is arbitrary amino acid residue, " Xao " is the amino acid residue for preferably being selected from Glu, Gln or Ser, and " Xap " is the amino acid residue for preferably being selected from Thr or Val. The cutting sequence of Caspase-14 includes but is not limited to Leu Glu Xaa Asp (SEQ ID NO:34), wherein " Xaa " is any Amino acid residue.The cutting sequence of complement pathway C3/C5 invertase includes but is not limited to Gln Leu Gly Arg Leu His Met Lys (SEQ ID NO:35) (cleavage site is between Arg and Leu) and Gly Leu Ala Arg Ser Asn Leu Asp (SEQ ID NO:36) (cleavage site is between Arg and Ser).The cutting sequence of MMP-8 includes but is not limited to Gly Xaq Xaa Gly (SEQ ID NO:37), wherein " Xaa " is arbitrary amino acid residue, and " Xaq " is preferably to be selected from Pro, Ala or Ser Amino acid residue.The cutting sequence of MMP-11 includes but is not limited to Xaa Ala Ala Ala (SEQ ID NO:38), wherein " Xaa " is arbitrary amino acid residue.The cutting sequence of MMP-12 include but is not limited to Gly Xar Xas Xas (SEQ ID NO: 39), wherein " Xar " is the amino acid residue for preferably being selected from Pro, Ala or Gly, " Xas " is preferably to be selected from Ala or the amino acid of Gly is residual Base.The cutting sequence of MMP-13 includes but is not limited to that (cleavage site exists Gly Pro Xaa Gly Xat (SEQ ID NO:40) Between Gly and Xat), wherein " Xaa " be arbitrary amino acid residue, " Xat " be preferably be selected from Leu, Ile or Val amino acid it is residual Base, preferably Gly Pro Ala Gly Leu (SEQ ID NO:41).The cutting sequence of MMP-20 includes but is not limited to Pro Xaa Leu Pro Xau (SEQ ID NO:42) (cleavage site is between Pro and Xau), wherein " Xaa " is arbitrary amino acid residue, " Xau " is the amino acid residue for preferably being selected from Leu or Met, preferably Pro Ala Leu Pro Leu (SEQ ID NO:43) or Pro Ala Leu Pro Met(SEQ ID NO:44)。
In some embodiments, the phenylalanine oxidizing ferment proenzyme sequence is as shown in SEQ ID NO:1.
In some embodiments, modified phenylalanine oxidizing ferment proenzyme sequence such as SEQ ID NO:2,4-7 Described in any one.
In second aspect, the present invention provides modified phenylalanine oxidizing ferment proenzymes described in first aspect for detecting The purposes of proteinase activity in sample.
In the present invention, the sample can be body fluid, whole blood, blood plasma, serum or tissue.
In prothrombin a Activity determination, to derive from the phenylalanine oxidizing ferment of Pseudomonas Sp.P-501 Proenzyme is reaction substrate, by technique for gene engineering in the specific recognition sequence for wherein introducing prothrombin a, utilizes phenylpropyl alcohol ammonia The redox and chromogenic reaction of acid oxidase (PAO) and substrate, building prothrombin a concentration and reaction product absorbance become Time graph between change establishes the active direct detection architecture of prothrombin a.Meanwhile using clinical sample to detection architecture Feasibility verifying is carried out, confirmation detection method is suitable for the detection of the anticoagulant plasma sample of sodium citrate.The present invention constructs completely Prothrombin a detection method, by prothrombin a, oxidoreducing enzyme two-stage cascade react, widened detection range, Improve detection sensitivity.
In the embodiment of low content testing protein enzyme, such as factor Xa, need to increase with the detection architecture Add a step activation of zymogen cascade reaction to amplify the concentration signal of Xa factor, such as middle protein proenzyme meets basic amplification and wants It asks, that is, is not present in human body, identification sequence-specific is strong, and pH value in reaction is in neutrality, and can detect blood plasma in 30 minutes The absorbance change of sample.
In some embodiments, middle protein proenzyme is modified NisP protease zymogens (proNisP), preceding It leads between peptide and catalytic subunit comprising enzyme spcificity recognition site sequence.For example, modified proNisP comes from nisin Bacterium.
During the Activity determination of factor Xa and IIa, blood plasma is pre-processed with streptokinase, can be prevented complete The solidification of plasma sample when state of activation.
In the detection of fibrinolysin enzymatic activity, plasminogen can directly be activated by streptokinase generates fibrinolysin, Hou Zheyin Enzyme-linked activating reaction is played, to establish the direct detection architecture of plasmin activity.
In addition, feasibility verifying has been carried out to coagulation factor and fibrinolysin using sodium citrate anticoagulant plasma sample, than Compared with each proteinase activity of the blood plasma under physiological status and full activation state.
In the detection of Caspase-3 enzymatic activity, using phenylalanine oxidizing ferment proenzyme as reaction substrate, pass through genetic engineering Technology is built into the specific recognition sequence of Caspase-3, using the redox and chromogenic reaction of PAO and substrate, establishes The active direct detection architecture of Caspase-3.Meanwhile feasibility verifying, confirmation inspection are carried out to detection architecture using clinical sample Survey method is suitable for the detection of serum sample.
In the detection of C5 converting Enzyme enzymatic activity, using phenylalanine oxidizing ferment proenzyme as reaction substrate, pass through genetic engineering Technology is built into the specific recognition sequence of C5 converting Enzyme, using the redox and chromogenic reaction of PAO and substrate, establishes complement system The active direct detection architecture of C5 converting Enzyme in system.Meanwhile feasibility verifying, confirmation are carried out to detection architecture using clinical sample Detection method is suitable for the detection of serum sample.
In some embodiments, the plasma specimen used is the anticoagulant plasma sample of sodium citrate or Heparin plasma sample This.
It in some embodiments, further include using streptokinase or urokinase pretreatment sample.
In some embodiments, the redox and chromogenic reaction of the phenylalanine oxidation enzyme-to-substrate including activation.
In the third aspect, the present invention provides a kind of isolated nucleic acid, encode modified benzene described in first aspect Alanine oxidizing ferment proenzyme.
In fourth aspect, the present invention provides a kind of expression vectors, and it includes the isolated nucleic acid described in the third aspect.
At the 5th aspect, the present invention provides the host cells of the conversion of the expression vector described in fourth aspect or transfection.
6th aspect, the present invention provides a kind of kits of proteinase activity in test sample comprising first aspect The modified phenylalanine oxidizing ferment proenzyme.
In some embodiments, the kit also includes modified proNisP, in leader peptide and catalytic subunit Between include enzyme spcificity recognition site sequence, such as the specific recognition sequence of factor Xa.
The L-phenylalanine oxidizing ferment proenzyme (proPAO) of not catalytic activity from Psuedomonas Sp.P-501 Substantially by the leader sequence of 14 amino acid residues, the α subunit of 92 amino acid residues, a dipeptides (Ile-Lys) and 605 The β subunit of residue forms.According to the experimental result of protease hydrolytic proPAO external in existing document, proPAO can be by albumen α subunit and β subunit fragments of enzyme Pronase and the Trypsin cohydrolysis at first lysine residue of missing, the hydrolysate Activity (Suzuki, H.et al.Sequencing suitable with the PAO activity of the activated form containing α subunit and β subunit fragments and expression of the L-phenylalanine oxidase gene from Pseudomonas sp.P- 501.Proteolytic activation of the proenzyme.Journal of biochemistry 136,617- 627,doi:10.1093/jb/mvh169(2004)；Ida,K.et al.Structural basis of proteolytic activation of L-phenylalanine oxidase from Pseudomonas sp.P-501.The Journal of biological chemistry 283,16584-16590,doi:10.1074/jbc.M800366200(2008)；Ida, K.,Suguro,M.&Suzuki,H.High resolution X-ray crystal structures of L- phenylalanine oxidase(deaminating and decarboxylating)from Pseudomonas sp.P- 501.Structures of the enzyme-ligand complex and catalytic mechanism.Journal of biochemistry 150,659-669,doi:10.1093/jb/mvr103(2011)).Therefore, α referred to herein is sub- Base includes the form that N-terminal lacks a lysine, and alleged leader sequence then can an accordingly bad ammonia more than the C-terminal at this time Acid.
Definition
Phenylalanine oxidizing ferment: referring to phenylalanine 2- monooxygenase (EC 1.13.12.9), major catalytic L- phenylpropyl alcohol ammonia Acid generates 2- phenyl-acetamides or 3- phenylpyruvic acid, substrate further include β -2- thienylalanine, l-tyrosine, l-methionine Deng.
Proenzyme: the precursor forms of the enzyme of the given activity with enzyme, the thus commonly referred to as enzyme of inactive form.Herein So-called proenzyme can refer to the precursor forms in detection method through digestion activation at active enzyme.Proenzyme can be by causing to live Change other cascade enzyme activations and answers organized enzyme in pairs.
Modification: the mutation (displacement) of one or more amino acid residues in polypeptide (such as enzyme), be inserted into (addition) or Missing, to obtain the polypeptide of modification.Herein, term " recombination ", " modification ", " transformation " are used interchangeably.
Recognition site sequence: refer in detection by enzyme institute's specific recognition and the amino acid sequence of cutting.Herein, term " recognition site sequence ", " identification sequence " or " cutting sequence " is used interchangeably.
Protease zymogens: the protease precursor form of Viability protease can be activated.Herein referred protease includes silk Serine protease, serine/threonine protein enzyme, cysteine proteinase, aspartic protease, metalloproteinases or hydroxyproline The enzyme of any cleavable protein sequence such as enzyme.
Amino acid residue: including but is not limited to the amino acid residue for the set being made of following amino acid: alanine (three words Female code: Ala, single letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), half Guang ammonia Sour (Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y) and valine (Val, V)。
Coded sequence: the boundary of coded sequence is generally determined that the open reading frame is close from originating by open reading frame Numeral (such as ATG, GTG or TTG) starts and with terminator codon (such as TAA, TAG or TGA) end.Coded sequence can be base Because of a group DNA, cDNA, synthetic DNA or combinations thereof.
Conversion: referring to DNA importing recipient host cell, changes genotype and subsequently results in the change in recipient cell.
Host cell: refer to by using recombinant DNA technology building and encoding what the carrier of at least one heterologous gene converted Cell.
Method for detecting enzymatic activity:
A. prothrombin a
1. prothrombin a specific substrate (includes prothrombin a specific recognition sites sequence through modifying phenylpropyl alcohol ammonia Acid oxidase proenzyme) preparation method
The preparation method of prothrombin a specific substrate, including bacterial strain, plasmid, enzyme and culture medium, PCR amplification and recombination The building of plasmid, the inducing expression of gene and the purifying of protein.Specific step is as follows:
(1) bacterial strain, plasmid, enzyme and culture medium:
Escherichia coli (Escherichia coli) DH5 α, BL21 (DE3), cloning and expression plasmid vector pRSFDuet-1 Purchased from Novagen company；DNA high fidelity polymerase enzyme mixation is purchased from Tsingke company；Restriction enzyme, DNA ligase purchase From NEB company；Escherichia coli LB culture medium: every liter of 10g containing Tryptone, Yeast extract 5g, NaCl 10g block that Mycin concentration is 50mg/L.
(2) Overlap extension PCR amplification and the building of recombinant plasmid:
The gene order of full genome composite coding proPAO (i.e. SEQ ID NO:1), designs three pairs of primers, is prolonged by overlapping Stretch PCR be added (i.e. between leader sequence and α subunit between α subunit and β subunit) at the activation site of proenzyme proPAO two it is solidifying The specific recognition sequence (amino acid sequence LRPR, SEQ ID NO:9) of blood factor IIa.It is connected through restriction enzymes double zyme cutting To the expression vector pRSFDuet-1 through same endonuclease digestion, connection product converts E. coli DH5 α, screening Recombinant plasmid, and carry out double digestion and sequence verification.
The primer of above-mentioned Overlap extension PCR is as follows:
Upstream PAO A1-45:
5′-catgccatggtgggcgttaccgtcattccccggctgc-3′(SEQ ID NO:45)
Downstream PAO A2-46:
5′-gcccgcgtaccttgatcgcacgtggacgcagtcccgggcggtcatgaaga-3′(SEQ ID NO: 46)
Upstream PAO B1-47:
5′-tcttcatgaccgcccgggactgcgtccacgtgcgatcaaggtacgcgggc-3′(SEQ ID NO: 47)
Downstream PAO B2-48:
5′-ggaattccatatgttaatgatgatgatgatgatgctggctggtggccagctccgc-3′(SEQ ID NO:48)
Upstream PAO C1-49:
5′-cggcctgaaggacgagaagctgcgtccacgtaagattgccaccaccgttg-3′(SEQ ID NO: 49)
Downstream PAO C2-50:
5′-caacggtggtggcaatcttacgtggacgcagcttctcgtccttcaggccg-3′(SEQ ID NO: 50)
Wherein, dashed part is the proPAO upstream primer NcoI restriction enzyme site after modification and downstream primer NdeI digestion position Point.Restriction enzyme selects NcoI and two kinds of NdeI.
(3) purifying of the inducing expression of gene and protein:
By recombinant plasmid transformed E.coli BL21 (DE3).IPTG is added and carries out inducing expression in culture.Induce table The albumen reached passes through ultrasonication, and centrifugation takes supernatant to carry out affinity chromatography (QIAGEN Products) and desalination chromatography The proPAO for the prothrombin a substrate specificity that column (Amersham Products) is purified.
(4) prothrombin a digests the verifying of substrate
Commercialization prothrombin a sterling (Sigma Products) is taken, is added to the bottom prothrombin a of above-mentioned purifying Object specificity proPAO protein solution, 37 DEG C of incubations, SDS-PAGE verify hydrolysis efficiency.
2. the foundation of prothrombin a activity test method
(1) enzymatic hydrolysis of the prothrombin a to its substrate
After prothrombin a catalyzing hydrolysis, α is released prothrombin a substrate specificity proPAO with β subunit, The structure of PAO is unaffected, has catalytic activity.
(2) redox reaction and chromogenic reaction of PAO and substrate
Reaction process can be divided into two steps:
Firstly, the oxidative deamination of PAO catalysis substrate L-phenylalanine (L-Phe) reacts, β-phenylpyruvic acid (β-is generated Phenylpyruvate), ammonia (NH3) and hydrogen peroxide (H2O2).It is reacted then according to Trinder, in horseradish peroxidase (HRP) under catalytic action, hydrogen peroxide (H2O2) with amphyl (TOOS), 4-AA (4-AAT) react generation Quinones color-developing compounds (Quinones), the compound are reddish violet, and maximum absorption band is located at 550-560nm, color intensity It is directly proportional to the concentration of hydrogen peroxide.To eliminate the random error in detection process, the signal detection of this system chooses dual wavelength Spectrophotometry.It is measurement wavelength with wavelength 550-560nm at maximum absorption band, to absorb the wave at minimum in visible-range Long 700nm is combined wave length.With Abs555nm-Abs700nmHandle absorbance detection data, the analysis signal as detection.
(3) optimization of Enzyme assay condition
Enzyme reaction speed is accelerated with the raising of substrate proPAO concentration.However, the proPAO of high concentration will cause substrate Space length it is excessively close, lead to higher proPAO background response.Therefore the proPAO concentration for selecting reaction rate relatively high for Detect the final concentration of reaction.Other substrate (L-Phe, HRP, TOOS and 4-AAT) concentration and pH value in reaction are carried out respectively simultaneously Optimization, so that reaction system has maximum absorbance rate of change.
3. the detection of clinical samples
Using the anticoagulant plasma specimen of clinical sodium citrate, enough calcium ion (CaCl are supplemented in the reaction system2) and Tissue factor (Tissue Factor, TF) is sufficiently to activate the prothrombin a in test plasma.For the fibre in removal sample Fibrillarin original pre-processes sample to prevent sample from solidifying, using streptokinase (Streptokinase, SK) with fibre therein of degrading Fibrillarin, then processed sample is detected, it can prevent reaction system from solidifying.
B. factor Xa
In human body blood coagulation system, factor Xa is located at the upstream of prothrombin a, and it is intermediate to pass through selection in the present invention Proenzyme increases step activation of zymogen reaction to amplify the concentration signal of Xa factor.It has been specifically chosen from streptococcus lactis NisP enzyme proenzyme (the Nisin leader peptide-processing serine of (Streptococcus lactis) Protease proenzyme, proNisP).The proNisp gene order of full genome synthesis recombination, in the leader peptide of the proenzyme The specific recognition sequence IEGR (SEQ ID NO:17) of Xa factor is added between sequence and catalytic subunit.Then three pairs are designed to draw Specific recognition sequence ASPRI (the SEQ ID of two NisP is added by Overlap extension PCR at the activation site of proPAO for object NO:67)。
The primer of above-mentioned Overlap extension PCR is as follows:
Upstream PAO A1-45:
5′-catgccatggtgggcgttaccgtcattccccggctgc-3′(SEQ ID NO:45)
Downstream PAO A2-51:
5′-gcccgcgtaccttgatcgcaatacgaggagatgctcccgggcggtcatga-3′(SEQ ID NO: 51)
Upstream PAO B1-52:
5′-tcatgaccgcccgggagcatctcctcgtattgcgatcaaggtacgcgggc-3′(SEQ ID NO: 52)
Downstream PAO B2-48:
5′-ggaattccatatgttaatgatgatgatgatgatgctggctggtggccagctccgc-3′(SEQ ID NO:48)
Upstream PAO C1-53:
5′-cctgaaggacgagaaggcatctcctcgtattaagattgccaccaccgttg-3′(SEQ ID NO: 53)
Downstream PAO C2-54:
5′-caacggtggtggcaatcttaatacgaggagatgccttctcgtccttcagg-3′(SEQ ID NO: 54)
Wherein, dashed part is improved proPAO upstream primer NcoI restriction enzyme site and downstream primer NdeI digestion position Point.Restriction enzyme selects NcoI and two kinds of NdeI.
The recombination proenzyme proNisp and proPAO that build are respectively connected to expression vector pRSFDuet-1 and are transformed into E.coli BL21 (DE3) carries out the inducing expression of gene and the purifying of protein respectively.The inducing expression and albumen of proNisp The purifying of matter is as follows: IPTG is added and carries out inducing expression in culture.The albumen of inducing expression passes through ultrasonication, is centrifuged, Supernatant progress affinity chromatography (QIAGEN Products) and desalination chromatographic column (Amersham Products) is taken to obtain egg White product.
Commercialization factor Xa sterling (NEB Products) and the recombination proNisP proenzyme of purifying is taken to be added to above-mentioned Recombination proPAO, 37 DEG C of incubations, SDS-PAGE verifies hydrolysis efficiency.
The factor Xa Activity determination of clinical samples: using the anticoagulant plasma specimen of clinical sodium citrate, in reactant Enough CaCl are supplemented in system2With TF sufficiently to activate the factor Xa in test plasma.For the fiber egg in removal sample White original uses streptokinase to pre-process sample with fibrin therein of degrading, then to processed mark to prevent sample from solidifying This is detected.
C. fibrinolysin
Using proPAO (Pseudomonas Sp.P-501) gene, as template, three pairs of primers is designed, Overlap extension PCR is passed through The specific recognition sequence GYRA (SEQ ID NO:23) of two Plasmin is added at the activation site of proenzyme proPAO.Through limiting Property enzymes double zyme cutting processed is connected on the expression vector pRSFDuet-1 through same endonuclease digestion, and connection product converts large intestine Bacillus E.coli DH5 α screens recombinant plasmid, and carries out double digestion and sequence verification.
The primer of above-mentioned Overlap extension PCR is as follows:
Upstream PAO A1-45:
5′-catgccatggtgggcgttaccgtcattccccggctgc-3′(SEQ ID NO:45)
Downstream PAO A2-55:
5′-cagcccgcgtaccttgatcgcacgataacctcccgggcggtcatgaagaa-3′(SEQ ID NO: 55)
Upstream PAO B1-56:
5′-ttcttcatgaccgcccgggaggttatcgtgcgatcaaggtacgcgggctg-3′(SEQ ID NO: 56)
Downstream PAO B2-48:
5′-ggaattccatatgttaatgatgatgatgatgatgctggctggtggccagctccgc-3′(SEQ ID NO:48)
Upstream PAO C1-57:
5′-cggcctgaaggacgagaagggttatcgtgcaaagattgccaccaccgttg-3′(SEQ ID NO: 57)
Downstream PAO C2-58:
5′-caacggtggtggcaatctttgcacgataacccttctcgtccttcaggccg-3′(SEQ ID NO: 58)
Wherein, dashed part is improved proPAO upstream primer NcoI restriction enzyme site and downstream primer NdeI digestion position Point.Restriction enzyme selects NcoI and two kinds of NdeI.
Subsequent implementation content is referred to prothrombin a activity test method, it may be noted that with prothrombin a and Unlike Xa, in the embodiment of plasmin activity detection method, streptokinase can directly activate the fibrinolysin in blood plasma Original is allowed to generate fibrinolysin, and the latter causes enzyme-linked activating reaction, to establish the direct detection architecture of plasmin activity.
D.Caspase-3
Using proPAO (Pseudomonas Sp.P-501) gene, as template, three pairs of primers is designed, Overlap extension PCR is passed through The specific recognition sequence DEVD (SEQ ID NO:27) of two Caspase-3 is added at the activation site of proenzyme proPAO.Through Restriction enzymes double zyme cutting is connected on the expression vector pRSFDuet-1 through same endonuclease digestion, and connection product conversion is big Enterobacteria E.coli DH5 α screens recombinant plasmid, and carries out double digestion and sequence verification.
The primer of above-mentioned Overlap extension PCR is as follows:
Upstream PAO A1-45:
5′-catgccatggtgggcgttaccgtcattccccggctgc-3′(SEQ ID NO:45)
Downstream PAO A2-59:
5′-gcccgcgtaccttgatcgcatcaacttcgtctcccgggcggtcatgaaga-3′(SEQ ID NO: 59)
Upstream PAO B1-60:
5′-tcttcatgaccgcccgggagacgaagttgatgcgatcaaggtacgcgggc-3′(SEQ ID NO: 60)
Downstream PAO B2-48:
5′-ggaattccatatgttaatgatgatgatgatgatgctggctggtggccagctccgc-3′(SEQ ID NO:48)
Upstream PAO C1-61:
5′-cggcctgaaggacgagaaggacgaagttgataagattgccaccaccgttg-3′(SEQ ID NO: 61)
Downstream PAO C2-62:
5′-caacggtggtggcaatcttatcaacttcgtccttctcgtccttcaggccg-3′(SEQ ID NO: 62)
Wherein, dashed part is improved proPAO upstream primer NcoI restriction enzyme site and downstream primer NdeI digestion position Point.Restriction enzyme selects NcoI and two kinds of NdeI.
The enzyme activity unit of Caspase-3: Caspase-3 hydrolyzable acetyl group-Asp-Glu-Val-Asp- p-nitrophenyl Amine (Ac-DEVD-pNA) discharges the part paranitroanilinum (pNA), can measure at 405nm.By detecting recombinant C aspase-3 The enzyme activity unit of Caspase-3 can be defined to the cleavage activity of chromogenic substrate Ac-DEVD-pNA.
The implementation content of subsequent substrate proPAO activating reaction is referred to prothrombin a activity test method, needs to infuse Meaning, unlike the detection method in blood coagulation system, in the embodiment of all Caspase activity test methods, is adopted Clinical samples are serum composition.In addition, do not need in reaction using activator, it can be directly by detection serum It is active directly to establish Caspase-3 to the activating reaction of the recombination proenzyme proPAO containing special cutting sequence by Caspase-3 Detection architecture.
E.C5 invertase
Using proPAO (Pseudomonas Sp.P-501) gene, as template, three pairs of primers is designed, Overlap extension PCR is passed through The specific recognition sequence QLGRLHMK (SEQ ID NO:35) of two C5 convertases is added at the activation site of proenzyme proPAO. It is connected on the expression vector pRSFDuet-1 through same endonuclease digestion through restriction enzymes double zyme cutting, connection product conversion E. coli DH5 α screens recombinant plasmid, and carries out double digestion and sequence verification.
The primer of above-mentioned Overlap extension PCR is as follows:
Upstream PAO A1-45:
5′-catgccatggtgggcgttaccgtcattccccggctgc-3′(SEQ ID NO:45)
Downstream PAO A2-63:
5′-cgcgtaccttgatcgctttcatgtgcagacgacccagttgtcccgggcggtcatga-3′(SEQ ID NO:63)
Upstream PAO B1-64:
5′-tcatgaccgcccgggacaactgggtcgtctgcacatgaaagcgatcaaggtacgcg-3′(SEQ ID NO:64)
Downstream PAO B2-48:
5′-ggaattccatatgttaatgatgatgatgatgatgctggctggtggccagctccgc-3′(SEQ ID NO:48)
Upstream PAO C1-65:
5′-cctgaaggacgagaagcaactgggtcgtctgcacatgaagattgccaccaccgttg-3′(SEQ ID NO:65)
Downstream PAO C2-66:
5′-caacggtggtggcaatcttcatgtgcagacgacccagttgcttctcgtccttcagg-3′(SEQ ID NO:66)
Wherein, dashed part is improved proPAO upstream primer NcoI restriction enzyme site and downstream primer NdeI digestion position Point.Restriction enzyme selects NcoI and two kinds of NdeI.
Subsequent implementation content is referred to Caspase-3 activity test method, can directly pass through C5 in detection serum Invertase establishes C5 convertase activity in complement system to the activating reaction of the recombination proenzyme proPAO containing special cutting sequence Direct detection architecture.
Detailed description of the invention
Fig. 1 is the SDS-PAGE detection of prothrombin a specific substrate proPAO (identifying sequence containing LRPR) purified product As a result, in which: M is albumen marker.
Fig. 2 is that prothrombin a detects knot to the SDS-PAGE of specific substrate proPAO (identifying sequence containing LRPR) enzymatic hydrolysis Fruit, in which: M is albumen marker.
Fig. 3 is enzymolysis activity of the prothrombin a sterling to factor IIa specific substrate proPAO (identifying sequence containing LRPR) Quantitative detection result.
Linear relationship of the Fig. 4 between prothrombin a unit of activity and corresponding reaction rate equation parameter.
Fig. 5 is the prothrombin a in blood plasma after streptokinase pretreatment to substrate proPAO's (identifying sequence containing LRPR) Enzymolysis activity testing result.
Fig. 6 is containing there are two the proNisP of factor Xa cleavage site (identifying sequence containing IEGR) and NisP specificity The SDS-PAGE testing result of substrate proPAO (identifying sequence containing ASPRI) purified product, in which: M is albumen marker.
Fig. 7 is that factor Xa digests the product NisP after factor Xa specific substrate proNisP (identifying sequence containing IEGR), And product NisP is to the SDS-PAGE testing result of NisP specific substrate proPAO (identifying sequence containing ASPRI) enzymatic hydrolysis, in which: M is albumen marker.
Fig. 8 is that the factor Xa after streptokinase pre-processes in blood plasma (is contained by digesting factor Xa specific substrate proNisP IEGR identifies sequence) to the enzyme-linked hydrolysing activity testing result of NisP specific substrate proPAO (identifying sequence containing ASPRI).
Fig. 9 (identifies sequence containing GYRA to specific substrate proPAO for the fibrinolysin in blood plasma after streptokinase plasminogen activation Column) enzymolysis activity testing result.
Figure 10 is to recombinate Caspase-3 to the cleavage activity testing result of synthesis substrate A c-DEVD-pNA.
Figure 11 is Caspase-3 in different subject's serum to the enzymatic hydrolysis of specific substrate proPAO (identifying sequence containing DEVD) Activity determination result.Wherein: Sample 1 is Small Cell Lung Cancer and Patients with Liver Metastasis, and Sample 2 is cardia lesion patient, Sample 3 is doubtful Lymphoma, and Sample 4 is examinee.
Figure 12 is that C5Convertase (identifies sequence containing QLGRLHMK to specific substrate proPAO in different subject's serum Column) enzymolysis activity testing result.Wherein: Sample 1 and Sample 2 are rheumatism immunological diseases patient, Sample 3 with Sample 4 is examinee.
A and B is respectively specific substrate polyphenol oxidase proenzyme proCPO (signal crayfish in Figure 13 Pacifastacus leniusculus) and the inspection that is activated by coagulation factor of proDPO (Drosophila melanogaster) Survey result.
Figure 14 is that the prothrombin a after streptokinase pre-processes in blood plasma (identifies sequence containing a LRPR to substrate proPAO Column) enzymolysis activity testing result.
Figure 15 is the prothrombin a in blood plasma after streptokinase pretreatment to from staphylococcus aureus The SplB protease specific substrate proPAO of (Staphylococcus aureus) (contains WELQ, SEQ ID NO:68 identifies sequence Column) enzymolysis activity testing result.
Figure 16 is the corresponding protease activity of activation of zymogen reaction assay that the proPAO of sequence modification is identified through specific proteases The flow chart of property.
Specific embodiment
The specific embodiment of proteinase activity detection includes coagulation cascade protease (including a variety of coagulation factors), fibrinolytic Enzyme, the aspartic protease (including Caspase family member) containing cysteine, complement pathway protein enzyme (includes each benefit Body ingredient) and matrix metalloproteinase (including MMP family member).
1. prothrombin a Activity determination of embodiment
1. the building of the proPAO sequence of prothrombin a substrate specificity
(1) amplification obtains the proPAO DNA fragmentation containing a prothrombin a cleavage site
The primer upstream PAO A1-45 and the downstream PAO A2-46 are separately designed, under the upstream PAO B1-47 and PAO B2-48 It swims, has been separately added into NcoI and NdeI restriction enzyme site in the primer upstream PAO A1-45 and the downstream PAO B2-48.Full genome synthesis ProPAO sequence (coded sequence is SEQ ID NO:1) in Pseudomonas Sp.P-501, using this DNA sequence dna as template, With the primer upstream PAO A1-45 and the downstream PAO A2-46, the upstream PAO B1-47 and the downstream PAO B2-48 expand contain one respectively Two parts DNA fragmentation A and B of a prothrombin a cleavage site (between α subunit and β subunit).PCR reaction system: 1.1 × PCR mix buffer, 20 μ l, upstream, downstream primer (10 μM) each 1 μ l, 0.2 μ l of template.Reaction condition: 94 DEG C of thermal changes Property 5min；94 DEG C of denaturation 45s, 60 DEG C of annealing 45s, 72 DEG C of extension 30s (when using primer PAO A1 and PAO A2) or 2min (make When with primer PAO B1 and PAO B2), totally 30 recycle；72 DEG C of extension 10min.The agar that PCR product is prepared in 0.5 × TAE Electrophoresis in sugared gel, DNA QIAquick Gel Extraction Kit (Tiangen company) recycle target fragment, and method refers to its specification, and recycling produces - 20 DEG C of object save backup.
By the A segment for using primer PAO A1-45 and PAO A2-46 to expand and use primer PAO B1-47 and PAO The B segment that B2-48 is obtained carries out Overlap extension PCR.Primer uses the upstream PAO A1-45 and the downstream PAO B2-48.PCR reaction System: 1.1 × PCR mix buffer, 20 μ l, upstream, downstream primer (10 μM) each 1 μ l, template A segment and each 1 μ l of B segment. Reaction condition: 94 DEG C of thermal denaturation 5min；94 DEG C of denaturation 45s, 60 DEG C of annealing 45s, 72 DEG C of extension 2min 10s, totally 30 recycle； 72 DEG C of extension 10min.Electrophoresis recycling PCR product is the same in Ago-Gel.The pure prothrombin a that contains is obtained to cut The DNA fragmentation in site (between α subunit and β subunit).
(2) the proPAO DNA fragmentation containing a prothrombin a cleavage site is cloned
Digestion with restriction enzyme is carried out to the obtained DNA fragmentation of recycling, reaction system: the PCR product of recycling 43 μ l, 10 × restriction enzyme enzyme buffer liquid 5 μ l, NcoI (Thermal Scientific company) 1 μ l, NdeI (Thermal Scientific company) 1 μ l.It mixes, 37 DEG C digest overnight.The PCR amplification gene Jing Guo digestion is recycled by agarose electrophoresis, It is connected with the plasmid vector pRSFDuet-1 recycled by same two digestion with restriction enzyme.Connect reactant System: the 12 μ l of PCR amplification gene handled by digestion, 2 μ l, 10 × T4 ligase buffer solution of plasmid vector handled by digestion 2 μ l, T4 ligase (Thermal Scientific company) 0.2 μ l, ddH2O 3.8μl.It mixes, 20 DEG C of reaction 2h.
The conversion of connection product: taking 100 μ l competent cell E.coli DH5 α in 1.5ml centrifuge tube, and 10 μ l are added and connect It practices midwifery object.30min is placed on ice, and 42 DEG C of heat shock 90s place 2min on ice.500 μ l LB culture mediums, 37 DEG C of shaken cultivations are added 45min makes bacteria resuscitation.
The culture of transformed bacteria: 12000rpm is centrifuged 1min, abandons supernatant, and thallus is resuspended with 100 μ l LB culture mediums, is coated on On the LB plate of kalamycin resistance.By plate in cultivating 16h in 37 DEG C of incubators.
The identification of recombinant plasmid: 10 μ l ddH are added in reaction tube in picking monoclonal colonies2O, which is resuspended, to be mixed, and 1 μ l is taken As template, bacterium colony PCR is carried out with primer PAO A1-45 and PAO B2-48.PCR result is carried out by agarose gel electrophoresis Identification.The correct monoclonal colonies of bacterium colony PCR are taken to be inoculated into the LB culture medium of 5ml kalamycin resistance, 37 DEG C of shaken cultivations 12h.Plasmid DNA is extracted in a small amount using plasmid extraction kit (Axygen company).Take 5 μ l plasmids, digestion with restriction enzyme, Digestion is confirmed by agarose gel electrophoresis as a result, selecting the correct recombinant plasmid of digestion identification, is selected corresponding primer, is sent Tsingke company carries out DNA sequencing analysis, confirms the correctness of objective gene sequence reading frame.
(3) amplification is obtained containing there are two the proPAO DNA fragmentations of prothrombin a cleavage site
The design primer upstream PAO C1-49 and the downstream PAO C2-50, respectively with the primer upstream PAO A1-45 and PAO C2- 50 downstreams, the upstream PAO C1-49 and the downstream PAO B2-48 are with the Plasmid DNA containing a prothrombin a cleavage site Template, amplification contains two parts DNA of second prothrombin a cleavage site (between leader peptide and α subunit) respectively Segment C and D.PCR reaction system and condition are the same as (1).The C segment that primer PAO A1-45 and PAO C2-50 will be used to expand Overlap extension PCR is carried out with the D segment for using primer PAO C1-49 and PAO B2-48 to obtain.Primer uses on PAO A1-45 Trip and the downstream PAO B2-48.PCR reaction system and condition are the same as (1).Obtain the pure prothrombin a cleavage site containing there are two ProPAO DNA fragmentation, corresponding amino acid sequence is shown in SEQ ID NO:2.
(4) clone is containing there are two the proPAO DNA fragmentations of prothrombin a cleavage site
Step is the same as (2).
2. the expression and purification of coagulation factor substrate specificity proPAO albumen
By aforementioned containing there are two the recombinant plasmid transformeds of the proPAO DNA fragmentation of the modification of prothrombin a cleavage site Enter E.coli BL21 (DE3).Monoclonal colonies on picking plate are inoculated into 5ml LB culture medium, 37 DEG C of shaken cultivations 12h.Above-mentioned whole cultures are inoculated into fresh LB containing 400ml, 37 DEG C of shaken cultivation 3h, as bacterium solution OD600nmIt reaches To 0.6, the IPTG of final concentration of 0.5mM is added into culture, 20 DEG C are continued to cultivate 16h.By culture be collected in 50ml from In heart pipe, 4 DEG C, 6000rpm is centrifuged 20min.Abandon supernatant.
Thallus dissolution: the bacterial sediment that centrifugation is obtained uses buffer solution A [50mM KH2PO4/K2HPO4, 300mM NaCl, pH8.0] sufficiently it is resuspended.
Bacterial cell disruption: selection ultrasonication.Ultrasonication condition is power 20%, ultrasonic 2s interval 4s, 5-30min.To After bacterium solution is limpid, in 4 DEG C, 13000rpm is centrifuged 30min, collects supernatant.
Affinity chromatography: the Ni-NTA suspension (QIAGEN company) of 0.8ml 50% is added into supernatant, in 4 DEG C of shaking tables At the uniform velocity mix 1h.Mixed liquor is added in chromatographic column, supernatant flows out under the effect of gravity, and Ni-NTA Stromal Precipitation is in chromatographic column In.The buffer solution A of 1 times of column volume is added into chromatographic column, washs chromatographic column, in triplicate.Liquid A stream to be buffered is net, to chromatography The buffer solution A that 5ml contains 20mM imidazoles is added in column, elutes impurity protein, in triplicate.Then, add by several times into chromatographic column Enter 2ml elution buffer B [50mM KH2PO4/K2HPO4, 300mM NaCl, 250mM Imidazole, pH8.0], elute purpose Albumen.Protein eluate is collected in 1.5ml centrifuge tube.
Desalting column chromatography and preservation: by obtained destination protein eluent in 4 DEG C, 13000rpm is centrifuged 30min, takes supernatant Liquid carries out desalination chromatography using ATKA protein purification instrument (Amersham company), and albumen is made to be in buffer C [10mM KH2PO4/ K2HPO4, pH8.0] in, glycerol is added to 10%, is saved backup in -80 DEG C.Quantification of protein kit is used simultaneously (Beyotime company) carries out assay to purified product, and destination protein sample is carried out SDS-PAGE electrophoresis to detect albumen Expression and purification situation.Electrophoretogram is as shown in Figure 1.
3. the enzymatic hydrolysis of prothrombin a is verified
The prothrombin a sterling (Sigma company) for taking people, by reaction system (1 μ g of prothrombin a sterling, modification 10 μ g, 30mM KH of proPAO2PO4/Na2HPO4, pH7.37), after 37 DEG C of incubation 3h, SDS-PAGE verifies hydrolysis efficiency.Electrophoresis Figure is as shown in Figure 2.Prothrombin a can successfully cut the proPAO of modification as seen from Figure 2.From left to right Each swimming lane be the modification that do not cut proPAO, the proPAO of modification is cut into have and smaller move by prothrombin a Prothrombin a, albumen marker is commercialized in the segment of shifting rate.
4. the foundation of prothrombin a activity test method
Trinder chromogenic reaction is selected, and optimizes corresponding chromogenic reaction concentration of substrate, buffer, reaction condition, is detected Condition determines the composition and Detection wavelength of two kinds of reaction reagents R1, R2, tentatively establishes detection architecture.
Reaction reagent R1:TOOS (N- ethyl-N- (2-Hydroxy-3-sulfopropyl)-3-methylaniline sodium salt)；Reaction reagent R2:L-Phe, HRP, 4-AAT.Absorbance detection reaction system: prothrombin a sterling (different unit of activity), coagulation factor IIa substrate specificity proPAO, reaction reagent R1, reaction reagent R2.Said components are added in ELISA Plate aperture, enzyme mark is used Instrument oscillation mixes reaction system, detects the absorbance at 555nm and 700nm and changes with time.
Data processing and drawing: △ Abs=Abs555nm-Abs700nm, using time t as abscissa, △ Abs is ordinate, is drawn Make the scatter plot of FIIa (Sigma company) sterling enzymolysis kinetics of different unit of activity.Testing result is as shown in Figure 3.By Fig. 3 It can be seen that with the increase of prothrombin a unit of activity in reaction system, the reaction rate that the proPAO of modification is cut It increases with it.
Function Fitting is carried out to describe corresponding reaction rate to each scatter plot respectively, is examined using simple linear regression dynamic Relationship between mechanical equation parameter and coagulation factor FIIa unit of activity.As a result as shown in Figure 4.It obtains as seen from Figure 4 It is linear related between kinetics equation parameter and FIIa unit of activity, R2=0.9989, explanation degree is good.
Optimizing detection reaction condition: the detection reagent containing different concentration of substrate is prepared respectively.Control other substrates and reaction Condition is constant, the reagent of various concentration of the selection containing same substrate, with the specific substrate after the hydrolysis of prothrombin a sterling It is reacted as sample.The absorbance change that reaction system is continuously monitored using microplate reader, using time t as abscissa, △ Abs For ordinate, scatter plot is drawn.Determining peak optimization reaction concentration of substrate is respectively 8mM TOOS, 7.5mM L- Phenylalanine, 6mM 4-AAT, 5U/ml HRP and 0.20 μ g/ μ l are modified proPAO, and optimal reaction temperature is 37 DEG C.
Optimizing detection pH value in reaction: under the conditions of the active optimal pH condition of prothrombin a and blood pH, gradient is prepared The phosphate buffer of pH.It is reacted using the specific substrate after the hydrolysis of prothrombin a sterling as sample, uses enzyme mark Instrument continuously monitors the absorbance change of reaction system.Using time t as abscissa, △ Abs is ordinate, draws scatter plot.Most preferably PH value is 7.35-7.45.
5. the Preliminary detection of clinical sample
(1) test experience of the anticoagulant plasma specimen of sodium citrate
Collect sample: choose in June, 2017 to Beijing Hospital's general out patient service during September (non-cardiovascular disease, it is non-pregnant, Be hospitalized reviewing patient, non-thyroid disorders etc. of non-orthopaedics have the patient of high thrombotic risk, what non-anticoagulant therapy and contraceptive used Patient) the anticoagulant plasma sample of sodium citrate, sodium citrate: (sodium citrate final concentration is about 0.109mmol/ to blood=1:9 L).To avoid random error to the greatest extent, plasma specimen is prepared into blood plasma disk with 10 parts for one group of mixing.It is with pooled plasma Sample is detected.
Sample detection: following ingredients (pooled plasma sample, reaction reagent R1 (90 μ l), reaction examination are added in ELISA Plate Agent R2 (90 μ l) and specific substrate proPAO (10 μ l)) after, be arranged the response procedures of microplate reader: oscillation mixes, detection 555nm and Absorbance changes with time at 700nm.
(2) plasma activator is tested
In the detection reaction system (proPAO of plasma sample, R1, R2 and modification) of the anticoagulant plasma specimen of sodium citrate CaCl is added2, while tissue factor (TF) is added, sufficiently to activate the prothrombin a in test plasma.
(3) streptokinase is tested
Preparation of samples: the standby anticoagulant pooled plasma sample of sodium citrate uses+100 μ l pooled plasma mark of 20 μ l streptokinase The pretreated plasma sample of this preparation.Check sample uses 20 μ l ddH2O+100 μ l pooled plasma sample is made.Clean Following ingredients (pretreatment sample/check sample (20 μ l), reaction reagent R1 (90 μ l), reaction reagent R2 (90 are added in ELISA Plate μ l) and modification proPAO (10 μ l)) after, use microplate reader carry out reaction detection.Testing result (caption box as shown in Figure 5 Middle substance is pre-mixed).After pre-processing blood plasma using streptokinase as seen from Figure 5, the prothrombin a of state of activation causes Enzyme-linked activating reaction.
(4) between fibrin ferment and absorbance quantitative relationship foundation
Trinder reaction is the common reaction of more mature biochemistry test, reaction condition, substrate and peroxide The selection of the concentration of enzyme all in accordance with peroxidase response parameter and obtain.For guarantee signal detection module reaction speed only with Concentration of hydrogen peroxide is directly proportional, and the concentration of remaining substrate and enzyme in the reaction system is much higher than KmValue.
Quantitative relation between absorbance value to signal detection and the concentration of sample to be tested prothrombin a is analyzed. It is linear between the absorbance value of signal detection and the concentration of sample to be tested prothrombin a at the first order reaction stage.Root It calculates, obtain absorbance value variation (△ Abs) and coagulates according to the time response curve of corresponding phenylalanine oxidizing ferment activation of zymogen Concentration (the c of blood factor IIa standard items(FIIa)) between equation y=ax+b, actually measured absorbance value is changed and is substituted into The concentration of prothrombin a in sample to be tested can be calculated in aforesaid equation.
2. factor Xa Activity determination of embodiment
Due to the activating reaction of a step middle protein proenzyme more than factor Xa Activity determination, first synthesize ProNisP (Streptococcus lactis) proenzyme DNA fragmentation of modification, the leader peptide sequences of the DNA fragmentation and catalysis are sub- It include the DNA sequence dna of the specific recognition sequence IEGR (SEQ ID NO:17) of factor Xa between basic sequence.Intact amino group Acid sequence figure is shown in SEQ ID NO:3.Digestion with restriction enzyme (BamHI, NcoI) is carried out to this DNA fragmentation, it is same as passing through The plasmid vector pRSFDuet-1 that recycles of two digestion with restriction enzyme be connected.Connection product is converted and reflected Surely the recombinant plasmid for obtaining correct sequence, by recombinant plasmid transformed E.coli BL21 (DE3).Monoclonal bacterium on picking plate Fall, carry out the inducing expression of modification albumen proNisP, affinity chromatography and desalination chromatography, the step of expression and purification referring to The expression and purification of prothrombin a substrate specificity proPAO.Destination protein sample is subjected to SDS-PAGE electrophoresis to detect The expression and purification situation of albumen.Electrophoresis is as shown in Figure 6.
Then, using the proPAO gene for encoding SEQ ID NO:1 as template, overlap-extension PCR is carried out with three pairs of corresponding primers PCR, it is final to obtain the DNA fragmentation containing two NisP cleavage sites, complete the structure of the enzyme-linked detection specific substrate sequence of Xa factor It builds, the amino acid sequence figure of coding is shown in SEQ ID NO:4.The expression and purification of the proPAO specific substrate albumen of modification referring to Corresponding description in prothrombin a embodiment.The SDS-PAGE electrophoresis of destination protein is as shown in Figure 6.
The enzymatic hydrolysis of factor Xa is verified: the factor Xa sterling (NEB company) of ox is taken, by reaction system (Xa factor 0.5 μ g of sterling, the 2 μ g of proNisP of modification, 6 μ g, the 30mM KH of proPAO of modification2PO4/Na2HPO4, pH 7.37), 37 DEG C After being incubated for 2.5h, SDS-PAGE verifies hydrolysis efficiency.Electrophoretogram is as shown in Figure 7.Factor Xa can be at as seen from Figure 7 The cutting to modification proPAO is realized to function by cutting modification proNisP.Each swimming lane from left to right is albumen marker, The modification proNisP not cut, cutting of the factor Xa to modification proNisP, factor Xa pass through cutting modification ProNisP the modification proPAO not cut, the modification proNisP not cut and repair to the cutting of modification proPAO Adorn proPAO, albumen marker.
The foundation of Xa factor activity test method: referring to prothrombin a activity test method, reaction system include Xa because Sub- sterling or the anticoagulant plasma sample of sodium citrate (20 μ l), the proNisP (6 μ l) of modification, the proPAO (20 μ l) of modification, reaction Reagent R1 (90 μ l), reaction reagent R2 (90 μ l).Said components are added in ELISA Plate aperture, are mixed using microplate reader oscillation anti- System is answered, the absorbance at 555nm and 700nm is detected and changes with time.
Data processing and drawing: △ Abs=Abs555nm-Abs700nm, using time t as abscissa, △ Abs is ordinate, is drawn The scatter plot of enzymolysis kinetics processed carries out Function Fitting to each scatter plot respectively to describe corresponding reaction rate.Sodium citrate The processing of anticoagulant plasma sample and Activiation method referring to prothrombin a Activity determination embodiment.Testing result such as Fig. 8 institute Show (substance is pre-mixed in caption box).After pre-processing blood plasma using streptokinase as seen from Figure 8, the blood coagulation of state of activation Factor Xa causes enzyme-linked activating reaction.
3. fibrinolysin Plasmin Activity determination of embodiment
Using the proPAO gene for encoding SEQ ID NO:1 as template, Overlap extension PCR is carried out with three pairs of corresponding primers, It is final to obtain the DNA fragmentation for containing two Plasmin cleavage sites (GYRA, SEQ ID NO:23), complete the inspection of Plasmin activity Survey the building of specific substrate sequence.Amino acid sequence figure is shown in SEQ ID NO:5.Modify proPAO specific substrate albumen expression with Purifying is referring to the corresponding description in prothrombin a embodiment.
The foundation of Plasmin activity test method: reaction system includes the anticoagulant plasma sample of sodium citrate (20 μ l), specifically The proPAO (5 μ l), reaction reagent R1 (90 μ l), reaction reagent R2 (90 μ l) of substrate modification.ELISA Plate is added in said components It in aperture, is vibrated using microplate reader and mixes reaction system, detected the absorbance at 555nm and 700nm and change with time.Data Embodiment of the processing with drawing referring to prothrombin a Activity determination.The fibre in blood plasma can be directly activated due to streptokinase Lyase original is allowed to generate fibrinolysin, and therefore, the activity of the Plasmin of full activation state can pass through the addition of streptokinase in blood plasma Directly it is detected.Testing result is as shown in Figure 9.It is raw after the Plasminogen in blood plasma is activated by streptokinase as seen from Figure 9 At Plasmin, the latter causes enzyme-linked activating reaction.
Embodiment 4.Caspase-3 Activity determination
Using the proPAO gene for encoding SEQ ID NO:1 as template, Overlap extension PCR is carried out with three pairs of corresponding primers, It is final to obtain the DNA fragmentation containing two Caspase-3 cleavage sites, complete Caspase-3 Activity determination specific substrate sequence Building.Amino acid sequence figure is shown in SEQ ID NO:6.Modify proPAO specific substrate albumen expression and purification referring to blood coagulation because Corresponding description in sub- IIa embodiment.
The activity test method of Caspase-3 albumen is as follows: by the chromogenic substrate of quantitative Caspase-3 and various concentration Ac-DEVD-pNA is added in ELISA Plate aperture, and reaction system includes Caspase-3 (2 μ l), Ac-DEVD-pNA (2 μ l), 30mM KH2PO4/Na2HPO4, pH8.0 (200 μ l), using microplate reader vibrate mix reaction system, detect 405nm at absorbance at any time Between variation.Testing result is as shown in Figure 10.As seen from Figure 10 under conditions of certain density Caspase-3, improve The concentration of substrate A c-DEVD-pNA can increase reaction rate.The corresponding enzyme of the available certain mass Caspase-3 of the reaction Unit of activity.
The foundation of Caspase-3 activity test method: reaction system includes serum sample (20 μ l), specific substrate modification ProPAO (5 μ l), reaction reagent R1 (90 μ l), reaction reagent R2 (90 μ l).Said components are added in ELISA Plate aperture, are used Microplate reader oscillation mixes reaction system, detects the absorbance at 555nm and 700nm and changes with time.Data processing and drawing Referring to the embodiment of prothrombin a Activity determination.The reaction can directly detect in serum Caspase-3 to containing The activating reaction of the modification proPAO of the special cutting sequence of Caspase-3.Testing result is as shown in figure 11.As seen from Figure 11 Caspase-3 contained in different serum specimens is different to the enzyme-linked activating reaction rate of modification proPAO.
The detection of embodiment 5.C5 invertase activity
Using the proPAO gene for encoding SEQ ID NO:1 as template, Overlap extension PCR is carried out with three pairs of corresponding primers, It is final to obtain the DNA fragmentation containing two C5 convertase cleavage sites, complete C5 convertase Activity determination specific substrate sequence Building.Amino acid sequence figure is shown in SEQ ID NO:7.The expression and purification of the protein modified proPAO of C5 convertase specific substrate referring to Corresponding description in prothrombin a embodiment.
The foundation of C5 convertase activity test method: reaction system includes serum sample (20 μ l), specific substrate modification ProPAO (15 μ l), reaction reagent R1 (90 μ l), reaction reagent R2 (90 μ l).Said components are added in ELISA Plate aperture, are made It is vibrated with microplate reader and mixes reaction system, detected the absorbance at 555nm and 700nm and change with time.Data processing with draw Figure referring to prothrombin a Activity determination embodiment.The reaction can directly detect in serum C5 convertase to containing should The activating reaction of the modification proPAO of the special cutting sequence of enzyme.Testing result is as shown in figure 12.Different blood as seen from Figure 12 C5 convertase contained in clear sample is different to the enzyme-linked activating reaction rate of modification proPAO.
Comparative example 6
In addition to phenylalanine oxidizing ferment proenzyme, the present invention has also selected other proenzymes and has carried out the transformation of specific substrate, For detecting the activity of protease in human body.In some embodiments, the polyphenol oxidase enzyme of two kinds of separate sources has been selected Original, respectively Prophenoloxidase (signal crayfish Pacifastacus leniusculus) are (referred to as ) and Prophenoloxidase 1 (Drosophila melanogaster) (referred to as proDPO) proCPO.Testing result is aobvious Show, corresponding protease just starts to show after about 100min to the activation of modification substrate proCPO, corresponding protease pair Substrate proDPO is modified almost without activation (as shown in figure 13).This is the result shows that both recombinate the sensitivity of proenzyme substrates Lower, detection time is longer, and unstable in blood plasma.
These comparative experiments show advantage of the phenylalanine oxidizing ferment proenzyme as substrates enzymes, it has specificity good, High sensitivity, detection time is short, facilitates a variety of advantages such as preparation.
Comparative example 7
Transformation position and particular sequence about phenylalanine oxidizing ferment proenzyme, the present invention in also carried out specific digestion Site sequence is only inserted between α subunit and β subunit, and test process is as follows: being to encode the proPAO gene of SEQ ID NO:1 Template carries out Overlap extension PCR with two pairs of corresponding primers, obtains the DNA fragmentation containing a prothrombin a cleavage site (between the α subunit and β subunit in phenylalanine oxidizing ferment proenzyme sequence) completes prothrombin a Activity determination specific substrate sequence The building of column.The activity test method of the expression and purification, prothrombin a of modifying proPAO is implemented referring to prothrombin a Corresponding description in scheme.Testing result is as shown in figure 14.As seen from Figure 14 in plasma sample prothrombin a activity It can hardly be detected.
As can be seen that dividing between the leader peptide sequences and α subunit of phenylalanine oxidizing ferment proenzyme, between α subunit and β subunit Not Han You specific restriction enzyme site when, the detection of certain proteinase activity can be completed.
Comparative example 8
For certain specific proteinase activated reaction, this kind of protease is not inserted into phenylalanine oxidizing ferment proenzyme When the cleavage sequences of preference, activation of zymogen reaction can not be detected.Test process is as follows: using proPAO gene as template, fortune Overlap extension PCR is carried out with three pairs of corresponding primers, obtains and derives from staphylococcus aureus (Staphylococcus containing one Aureus the DNA fragmentation of SplB proteolytic cleavage site (WELQ, SEQ ID NO:68)) completes the structure of specific substrate sequence It builds.The expression and purification of the protein modified proPAO of specific substrate, prothrombin a activity test method referring to coagulation factor Corresponding description in IIa embodiment.Testing result is as shown in figure 15.As seen from Figure 15 using without containing coagulation factor When the proPAO of IIa cleavage sequences, the activity of prothrombin a in plasma sample can not be detected, while anti-without colour developing It answers.Therefore in the implementation case, the corresponding special cutting sequence of protease is inserted into selection in phenylalanine oxidizing ferment proenzyme, is made Reaction, which must be detected, has the advantages such as speed is fast, sensitivity is high.
Sequence table
SEQ ID NO:1: the phenylalanine oxidizing ferment proenzyme amino acid sequence from Psuedomonas Sp.P-501
Leader peptide sequences show that α subunit is shown with dotted line underscore with runic double underline, and β subunit is with solid underline The precursor connector of display, connection α and β subunit is shown in bold.
SEQ ID NO:2: containing there are two the benzene from Psuedomonas Sp.P-501 of prothrombin a cleavage site The amino acid sequence (prothrombin a substrate specificity proPAO) of alanine oxidizing ferment proenzyme
Leader peptide sequences show that α subunit is shown with dotted line underscore with runic double underline, and β subunit is with solid underline Display.The cutting identification sequence LRPR of prothrombin a is located between leader peptide sequences and α subunit, α subunit and β subunit Between, it is shown with bold Italic.
SEQ ID NO:3: containing there are two factor Xa cleavage sites from streptococcus lactis (Streptococcus Lactis NisP enzyme proenzyme proNisP (factor Xa substrate specificity proNisP))
Leader peptide sequences are shown with double underline.The cutting identification sequence IEGR of factor Xa is located at leader peptide sequence Between column and catalytic subunit, shown with bold Italic.
SEQ ID NO:4: containing there are two the phenylalanines from Psuedomonas Sp.P-501 of NisP cleavage site Oxidizing ferment proenzyme (NisP substrate specificity proPAO)
Leader peptide sequences show that α subunit is shown with dotted line underscore with runic double underline, and β subunit is with solid underline Display.The identification cutting sequence ASPRI of NisP is located between leader peptide sequences and α subunit, between α subunit and β subunit, with Bold Italic is shown.
SEQ ID NO:5: containing there are two the phenylpropyl alcohol ammonia from Psuedomonas Sp.P-501 of Plasmin cleavage site Acid oxidase proenzyme (Plasmin specificity proPAO)
Leader peptide sequences show that α subunit is shown with dotted line underscore with runic double underline, and β subunit is with solid underline Display.The identification cutting sequence GYRA of Plasmin is located between leader peptide sequences and α subunit, between α subunit and β subunit, It is shown with bold Italic.
SEQ ID NO:6: containing there are two the phenylpropyl alcohols from Psuedomonas Sp.P-501 of Caspase-3 cleavage site Amino acid oxidase proenzyme (Caspase-3 substrate specificity proPAO)
Leader peptide sequences show that α subunit is shown with dotted line underscore with runic double underline, and β subunit is with solid underline Display.The identification cutting sequence DEVD of Caspase-3 is located between leader peptide sequences and α subunit, α subunit and β subunit it Between, it is shown with bold Italic.
SEQ ID NO:7: containing there are two the phenylpropyl alcohols from Psuedomonas Sp.P-501 of C5 convertase cleavage site Amino acid oxidase proenzyme (C5 convertase substrate specificity proPAO)
Leader peptide sequences show that α subunit is shown with dotted line underscore with runic double underline, and β subunit is with solid underline Display.The identification cutting sequence QLGRLHMK of C5 convertase is located between leader peptide sequences and α subunit, α subunit and β subunit Between, it is shown with bold Italic.
SEQ ID NO:8: prothrombin a specific cutting sequence
Xaa Xaa Xab Arg Xac
Wherein " Xaa " is arbitrary amino acid residue, and " Xab " is the amino acid residue for preferably being selected from Pro, Ala, Gly or Val, " Xac " is the amino acid residue for preferably being selected from Ser, Ala, Gly
SEQ ID NO:9: prothrombin a specific cutting sequence
Leu Arg Pro Arg
SEQ ID NO:10: prothrombin a specific cutting sequence
Leu Val Pro Arg Gly
SEQ ID NO:11: prothrombin a specific cutting sequence
Phe Pro Arg
SEQ ID NO:12: prothrombin a specific cutting sequence
Gly Arg Gly
SEQ ID NO:13: proconvertin a specific cutting sequence
Leu Ile Gln Arg
SEQ ID NO:14: the specific cutting sequence of factor IXa
Xaa Xaa Gly Arg
Wherein " Xaa " is arbitrary amino acid residue
SEQ ID NO:15: the specific cutting sequence of factor IXa
Pro Gln Gly Arg
SEQ ID NO:16: the specific cutting sequence of factor Xa
Xad Xaa Xae Xaf
Wherein " Xaa " is arbitrary amino acid residue, and " Xad " is the amino acid residue for preferably being selected from Ala or Ile, and " Xae " is excellent Amino acid residue selected from Pro, Phe or Gly, " Xaf " are the amino acid residue selected from Arg or Lys
SEQ ID NO:17: the specific cutting sequence of factor Xa
Ile Glu Gly Arg
SEQ ID NO:18: the specific cutting sequence of factor Xa
Ile Asp Gly Arg
SEQ ID NO:19: plasma thromboplastin antecedent a specific cutting sequence
Xag Xah Thr Arg
Wherein " Xag " is the amino acid residue for preferably being selected from Lys or Asp, and " Xah " is preferably to be selected from Phe or the amino acid of Leu is residual Base
SEQ ID NO:20: plasma thromboplastin antecedent a specific cutting sequence
Lys Leu Thr Arg
SEQ ID NO:21: Hageman factor a specific cutting sequence
Thr Ser Thr Arg
SEQ ID NO:22: fibrinolysin Plasmin specific cutting sequence
Xaa Xaa Xai Xaj
Wherein " Xaa " is arbitrary amino acid residue, and " Xai " is the amino acid residue for preferably being selected from Arg or Lys, and " Xaj " is excellent Amino acid residue selected from Ala, Ser, Gly or Arg
SEQ ID NO:23: fibrinolysin Plasmin specific cutting sequence
Gly Tyr Arg Ala
SEQ ID NO:24: fibrinolysin Plasmin specific cutting sequence
Pro Ala Lys Ala
The specific cutting sequence of SEQ ID NO:25:Caspase-2
Asp Glu Xaa Asp
Wherein " Xaa " is arbitrary amino acid residue
The specific cutting sequence of SEQ ID NO:26:Caspase-3
Xak Glu Val Asp
Wherein " Xak " is the amino acid residue for preferably being selected from Asp or Glu
The specific cutting sequence of SEQ ID NO:27:Caspase-3
Asp Glu Val Asp
The specific cutting sequence of SEQ ID NO:28:Caspase-6
Xaa Glu Xaa Asp
Wherein " Xaa " is arbitrary amino acid residue
The specific cutting sequence of SEQ ID NO:29:Caspase-7
Asp Xaa Xaa Asp
Wherein " Xaa " is arbitrary amino acid residue
The specific cutting sequence of SEQ ID NO:30:Caspase-8
Xal Xam Thr Asp
Wherein " Xal " is the amino acid residue for preferably being selected from Asp or Leu, and " Xam " is preferably to be selected from Glu or the amino acid of Ser is residual Base
The specific cutting sequence of SEQ ID NO:31:Caspase-8
Asp Glu Thr Asp
The specific cutting sequence of SEQ ID NO:32:Caspase-9
Xaa Xan Xaa Asp
Wherein " Xaa " is arbitrary amino acid residue, and " Xan " is the amino acid residue for preferably being selected from Asp or Glu
The specific cutting sequence of SEQ ID NO:33:Caspase-10
Xaa Xao Xap Asp
Wherein " Xaa " is arbitrary amino acid residue, and " Xao " is the amino acid residue for preferably being selected from Glu, Gln or Ser, " Xap " For the amino acid residue for preferably being selected from Thr or Val
The specific cutting sequence of SEQ ID NO:34:Caspase-14
Leu Glu Xaa Asp
Wherein " Xaa " is arbitrary amino acid residue
SEQ ID NO:35: the specific cutting sequence of complement pathway C3/C5 invertase
Gln Leu Gly Arg Leu His Met Lys
SEQ ID NO:36: the specific cutting sequence of complement pathway C3/C5 invertase
Gly Leu Ala Arg Ser Asn Leu Asp
The specific cutting sequence of SEQ ID NO:37:MMP-8
Gly Xaq Xaa Gly
Wherein " Xaa " is arbitrary amino acid residue, and " Xaq " is the amino acid residue for preferably being selected from Pro, Ala or Ser
The specific cutting sequence of SEQ ID NO:38:MMP-11
Xaa Ala Ala Ala
Wherein " Xaa " is arbitrary amino acid residue
The specific cutting sequence of SEQ ID NO:39:MMP-12
Gly Xar Xas Xas
Wherein " Xar " is the amino acid residue for preferably being selected from Pro, Ala or Gly, and " Xas " is the amino for preferably being selected from Ala or Gly Sour residue
The specific cutting sequence of SEQ ID NO:40:MMP-13
Gly Pro Xaa Gly Xat
Wherein " Xaa " is arbitrary amino acid residue, and " Xat " is the amino acid residue for preferably being selected from Leu, Ile or Val
The specific cutting sequence of SEQ ID NO:41:MMP-13
Gly Pro Ala Gly Leu
The specific cutting sequence of SEQ ID NO:42:MMP-20
Pro Xaa Leu Pro Xau
Wherein " Xaa " is arbitrary amino acid residue, and " Xau " is the amino acid residue for preferably being selected from Leu or Met
The specific cutting sequence of SEQ ID NO:43:MMP-20
Pro Ala Leu Pro Leu
The specific cutting sequence of SEQ ID NO:44:MMP-20
Pro Ala Leu Pro Met
SEQ ID NO:45: upstream PAO A1-45
5′-catgccatggtgggcgttaccgtcattccccggctgc-3′
SEQ ID NO:46: downstream PAO A2-46
5′-gcccgcgtaccttgatcgcacgtggacgcagtcccgggcggtcatgaaga-3′
SEQ ID NO:47: upstream PAO B1-47
5′-tcttcatgaccgcccgggactgcgtccacgtgcgatcaaggtacgcgggc-3′
SEQ ID NO:48: downstream PAO B2-48
5′-ggaattccatatgttaatgatgatgatgatgatgctggctggtggccagctccgc-3′
SEQ ID NO:49: upstream PAO C1-49
5′-cggcctgaaggacgagaagctgcgtccacgtaagattgccaccaccgttg-3′
SEQ ID NO:50: downstream PAO C2-50
5′-caacggtggtggcaatcttacgtggacgcagcttctcgtccttcaggccg-3′
SEQ ID NO:51: downstream PAO A2-51
5′-gcccgcgtaccttgatcgcaatacgaggagatgctcccgggcggtcatga-3′
SEQ ID NO:52: upstream PAO B1-52
5′-tcatgaccgcccgggagcatctcctcgtattgcgatcaaggtacgcgggc-3′
SEQ ID NO:53: upstream PAO C1-53
5′-cctgaaggacgagaaggcatctcctcgtattaagattgccaccaccgttg-3′
SEQ ID NO:54: downstream PAO C2-54
5′-caacggtggtggcaatcttaatacgaggagatgccttctcgtccttcagg-3′
SEQ ID NO:55: downstream PAO A2-55
5′-cagcccgcgtaccttgatcgcacgataacctcccgggcggtcatgaagaa-3′
SEQ ID NO:56: upstream PAO B1-56
5′-ttcttcatgaccgcccgggaggttatcgtgcgatcaaggtacgcgggctg-3′
SEQ ID NO:57: upstream PAO C1-57
5′-cggcctgaaggacgagaagggttatcgtgcaaagattgccaccaccgttg-3′
SEQ ID NO:58: downstream PAO C2-58
5′-caacggtggtggcaatctttgcacgataacccttctcgtccttcaggccg-3′
SEQ ID NO:59: downstream PAO A2-59
5′-gcccgcgtaccttgatcgcatcaacttcgtctcccgggcggtcatgaaga-3′
SEQ ID NO:60: upstream PAO B1-60
5′-tcttcatgaccgcccgggagacgaagttgatgcgatcaaggtacgcgggc-3′
SEQ ID NO:61: upstream PAO C1-61
5′-cggcctgaaggacgagaaggacgaagttgataagattgccaccaccgttg-3′
SEQ ID NO:62: downstream PAO C2-62
5′-caacggtggtggcaatcttatcaacttcgtccttctcgtccttcaggccg-3′
SEQ ID NO:63: downstream PAO A2-63
5′-cgcgtaccttgatcgctttcatgtgcagacgacccagttgtcccgggcggtcatga-3′
SEQ ID NO:64: upstream PAO B1-64
5′-tcatgaccgcccgggacaactgggtcgtctgcacatgaaagcgatcaaggtacgcg-3′
SEQ ID NO:65: upstream PAO C1-65
5′-cctgaaggacgagaagcaactgggtcgtctgcacatgaagattgccaccaccgttg-3′
SEQ ID NO:66: downstream PAO C2-66
5′-caacggtggtggcaatcttcatgtgcagacgacccagttgcttctcgtccttcagg-3′
The specific recognition sequence of SEQ ID NO:67:NisP
Ala Ser Pro Arg Ile
SEQ ID NO:68:SplB proteolytic cleavage site
Trp Glu Leu Gln
Sequence table
<110>Beijing Hospital
<120>modified phenylalanine oxidizing ferment proenzyme and application thereof
<130> LZ1811927CN01
<160> 68
<170> PatentIn version 3.3
<210> 1
<211> 721
<212> PRT
<213> Psuedomonas Sp.P501
<220>
<221>
<222>
<223>phenylalanine oxidizing ferment proenzyme amino acid
<400> 1
Met Val Gly Val Thr Val Ile Pro Arg Leu Leu Gly Leu Lys Asp Glu
1 5 10 15
Lys Lys Ile Ala Thr Thr Val Gly Glu Ala Arg Leu Ser Gly Ile Asn
20 25 30
Tyr Arg His Pro Asp Ser Ala Leu Val Ser Tyr Pro Val Ala Ala Ala
35 40 45
Ala Pro Leu Gly Arg Leu Pro Ala Gly Asn Tyr Arg Ile Ala Ile Val
50 55 60
Gly Gly Gly Ala Gly Gly Ile Ala Ala Leu Tyr Glu Leu Gly Arg Leu
65 70 75 80
Ala Ala Thr Leu Pro Ala Gly Ser Gly Ile Asp Val Gln Ile Tyr Glu
85 90 95
Ala Asp Pro Asp Ser Phe Leu His Asp Arg Pro Gly Ile Lys Ala Ile
100 105 110
Lys Val Arg Gly Leu Lys Ala Gly Arg Val Ser Ala Ala Leu Val His
115 120 125
Asn Gly Asp Pro Ala Ser Gly Asp Thr Ile Tyr Glu Val Gly Ala Met
130 135 140
Arg Phe Pro Glu Ile Ala Gly Leu Thr Trp His Tyr Ala Ser Ala Ala
145 150 155 160
Phe Gly Asp Ala Ala Pro Ile Lys Val Phe Pro Asn Pro Gly Lys Val
165 170 175
Pro Thr Glu Phe Val Phe Gly Asn Arg Val Asp Arg Tyr Val Gly Ser
180 185 190
Asp Pro Lys Asp Trp Glu Asp Pro Asp Ser Pro Thr Leu Lys Val Leu
195 200 205
Gly Val Val Ala Gly Gly Leu Val Gly Asn Pro Gln Gly Glu Asn Val
210 215 220
Ala Met Tyr Pro Ile Ala Asn Val Asp Pro Ala Lys Ile Ala Ala Ile
225 230 235 240
Leu Asn Ala Ala Thr Pro Pro Ala Asp Ala Leu Glu Arg Ile Gln Thr
245 250 255
Lys Tyr Trp Pro Glu Phe Ile Ala Gln Tyr Asp Gly Leu Thr Leu Gly
260 265 270
Ala Ala Val Arg Glu Ile Val Thr Val Ala Phe Glu Lys Gly Thr Leu
275 280 285
Pro Pro Val Asp Gly Val Leu Asp Val Asp Glu Ser Ile Ser Tyr Tyr
290 295 300
Val Glu Leu Phe Gly Arg Phe Gly Phe Gly Thr Gly Gly Phe Lys Pro
305 310 315 320
Leu Tyr Asn Ile Ser Leu Val Glu Met Met Arg Leu Ile Leu Trp Asp
325 330 335
Tyr Ser Asn Glu Tyr Thr Leu Pro Val Thr Glu Asn Val Glu Phe Ile
340 345 350
Arg Asn Leu Phe Leu Lys Ala Gln Asn Val Gly Ala Gly Lys Leu Val
355 360 365
Val Gln Val Arg Gln Glu Arg Val Ala Asn Ala Cys His Ser Gly Thr
370 375 380
Ala Ser Ala Arg Ala Gln Leu Leu Ser Tyr Asp Ser His Asn Ala Val
385 390 395 400
His Ser Glu Ala Tyr Asp Phe Val Ile Leu Ala Val Pro His Asp Gln
405 410 415
Leu Thr Pro Ile Val Ser Arg Ser Gly Phe Glu His Ala Ala Ser Gln
420 425 430
Asn Leu Gly Asp Ala Gly Leu Gly Leu Glu Thr His Thr Tyr Asn Gln
435 440 445
Val Tyr Pro Pro Leu Leu Leu Ser Asp Ser Ser Pro Ala Ala Asn Ala
450 455 460
Arg Ile Val Thr Ala Ile Gly Gln Leu His Met Ala Arg Ser Ser Lys
465 470 475 480
Val Phe Ala Thr Val Lys Thr Ala Ala Leu Asp Gln Pro Trp Val Pro
485 490 495
Gln Trp Arg Gly Glu Pro Ile Lys Ala Val Val Ser Asp Ser Gly Leu
500 505 510
Ala Ala Ser Tyr Val Val Pro Ser Pro Ile Val Glu Asp Gly Gln Ala
515 520 525
Pro Glu Tyr Ser Ser Leu Leu Ala Ser Tyr Thr Trp Glu Asp Asp Ser
530 535 540
Thr Arg Leu Arg His Asp Phe Gly Leu Tyr Pro Gln Asn Pro Ala Thr
545 550 555 560
Glu Thr Gly Thr Ala Asp Gly Met Tyr Arg Thr Met Val Asn Arg Ala
565 570 575
Tyr Arg Tyr Val Lys Tyr Ala Gly Ala Ser Asn Ala Gln Pro Trp Trp
580 585 590
Phe Tyr Gln Leu Leu Ala Glu Ala Arg Thr Ala Asp Arg Phe Val Phe
595 600 605
Asp Trp Thr Thr Asn Lys Thr Ala Gly Gly Phe Lys Leu Asp Met Thr
610 615 620
Gly Asp His His Gln Ser Asn Leu Cys Phe Arg Tyr His Thr His Ala
625 630 635 640
Leu Ala Ala Ser Leu Asp Asn Arg Phe Phe Ile Ala Ser Asp Ser Tyr
645 650 655
Ser His Leu Gly Gly Trp Leu Glu Gly Ala Phe Met Ser Ala Leu Asn
660 665 670
Ala Val Ala Gly Leu Ile Val Arg Ala Asn Arg Gly Asp Val Ser Ala
675 680 685
Leu Ser Thr Glu Ala Arg Pro Leu Val Ile Gly Leu Arg Pro Val Val
690 695 700
Lys Val Pro Ala Ala Glu Leu Ala Thr Ser Gln His His His His His
705 710 715 720
His
<210> 2
<211> 727
<212> PRT
<213>artificial (Artificial)
<220>
<221>
<222>
<223>prothrombin a substrate specificity proPAO
<400> 2
Met Val Gly Val Thr Val Ile Pro Arg Leu Leu Gly Leu Lys Asp Glu
1 5 10 15
Lys Leu Arg Pro Arg Lys Ile Ala Thr Thr Val Gly Glu Ala Arg Leu
20 25 30
Ser Gly Ile Asn Tyr Arg His Pro Asp Ser Ala Leu Val Ser Tyr Pro
35 40 45
Val Ala Ala Ala Ala Pro Leu Gly Arg Leu Pro Ala Gly Asn Tyr Arg
50 55 60
Ile Ala Ile Val Gly Gly Gly Ala Gly Gly Ile Ala Ala Leu Tyr Glu
65 70 75 80
Leu Gly Arg Leu Ala Ala Thr Leu Pro Ala Gly Ser Gly Ile Asp Val
85 90 95
Gln Ile Tyr Glu Ala Asp Pro Asp Ser Phe Leu His Asp Arg Pro Gly
100 105 110
Leu Arg Pro Arg Ala Ile Lys Val Arg Gly Leu Lys Ala Gly Arg Val
115 120 125
Ser Ala Ala Leu Val His Asn Gly Asp Pro Ala Ser Gly Asp Thr Ile
130 135 140
Tyr Glu Val Gly Ala Met Arg Phe Pro Glu Ile Ala Gly Leu Thr Trp
145 150 155 160
His Tyr Ala Ser Ala Ala Phe Gly Asp Ala Ala Pro Ile Lys Val Phe
165 170 175
Pro Asn Pro Gly Lys Val Pro Thr Glu Phe Val Phe Gly Asn Arg Val
180 185 190
Asp Arg Tyr Val Gly Ser Asp Pro Lys Asp Trp Glu Asp Pro Asp Ser
195 200 205
Pro Thr Leu Lys Val Leu Gly Val Val Ala Gly Gly Leu Val Gly Asn
210 215 220
Pro Gln Gly Glu Asn Val Ala Met Tyr Pro Ile Ala Asn Val Asp Pro
225 230 235 240
Ala Lys Ile Ala Ala Ile Leu Asn Ala Ala Thr Pro Pro Ala Asp Ala
245 250 255
Leu Glu Arg Ile Gln Thr Lys Tyr Trp Pro Glu Phe Ile Ala Gln Tyr
260 265 270
Asp Gly Leu Thr Leu Gly Ala Ala Val Arg Glu Ile Val Thr Val Ala
275 280 285
Phe Glu Lys Gly Thr Leu Pro Pro Val Asp Gly Val Leu Asp Val Asp
290 295 300
Glu Ser Ile Ser Tyr Tyr Val Glu Leu Phe Gly Arg Phe Gly Phe Gly
305 310 315 320
Thr Gly Gly Phe Lys Pro Leu Tyr Asn Ile Ser Leu Val Glu Met Met
325 330 335
Arg Leu Ile Leu Trp Asp Tyr Ser Asn Glu Tyr Thr Leu Pro Val Thr
340 345 350
Glu Asn Val Glu Phe Ile Arg Asn Leu Phe Leu Lys Ala Gln Asn Val
355 360 365
Gly Ala Gly Lys Leu Val Val Gln Val Arg Gln Glu Arg Val Ala Asn
370 375 380
Ala Cys His Ser Gly Thr Ala Ser Ala Arg Ala Gln Leu Leu Ser Tyr
385 390 395 400
Asp Ser His Asn Ala Val His Ser Glu Ala Tyr Asp Phe Val Ile Leu
405 410 415
Ala Val Pro His Asp Gln Leu Thr Pro Ile Val Ser Arg Ser Gly Phe
420 425 430
Glu His Ala Ala Ser Gln Asn Leu Gly Asp Ala Gly Leu Gly Leu Glu
435 440 445
Thr His Thr Tyr Asn Gln Val Tyr Pro Pro Leu Leu Leu Ser Asp Ser
450 455 460
Ser Pro Ala Ala Asn Ala Arg Ile Val Thr Ala Ile Gly Gln Leu His
465 470 475 480
Met Ala Arg Ser Ser Lys Val Phe Ala Thr Val Lys Thr Ala Ala Leu
485 490 495
Asp Gln Pro Trp Val Pro Gln Trp Arg Gly Glu Pro Ile Lys Ala Val
500 505 510
Val Ser Asp Ser Gly Leu Ala Ala Ser Tyr Val Val Pro Ser Pro Ile
515 520 525
Val Glu Asp Gly Gln Ala Pro Glu Tyr Ser Ser Leu Leu Ala Ser Tyr
530 535 540
Thr Trp Glu Asp Asp Ser Thr Arg Leu Arg His Asp Phe Gly Leu Tyr
545 550 555 560
Pro Gln Asn Pro Ala Thr Glu Thr Gly Thr Ala Asp Gly Met Tyr Arg
565 570 575
Thr Met Val Asn Arg Ala Tyr Arg Tyr Val Lys Tyr Ala Gly Ala Ser
580 585 590
Asn Ala Gln Pro Trp Trp Phe Tyr Gln Leu Leu Ala Glu Ala Arg Thr
595 600 605
Ala Asp Arg Phe Val Phe Asp Trp Thr Thr Asn Lys Thr Ala Gly Gly
610 615 620
Phe Lys Leu Asp Met Thr Gly Asp His His Gln Ser Asn Leu Cys Phe
625 630 635 640
Arg Tyr His Thr His Ala Leu Ala Ala Ser Leu Asp Asn Arg Phe Phe
645 650 655
Ile Ala Ser Asp Ser Tyr Ser His Leu Gly Gly Trp Leu Glu Gly Ala
660 665 670
Phe Met Ser Ala Leu Asn Ala Val Ala Gly Leu Ile Val Arg Ala Asn
675 680 685
Arg Gly Asp Val Ser Ala Leu Ser Thr Glu Ala Arg Pro Leu Val Ile
690 695 700
Gly Leu Arg Pro Val Val Lys Val Pro Ala Ala Glu Leu Ala Thr Ser
705 710 715 720
Gln His His His His His His
725
<210> 3
<211> 707
<212> PRT
<213>artificial (Artificial)
<220>
<221>
<222>
<223>factor Xa substrate specificity proNisP
<400> 3
Met Gly His His His His His His Gly Gly Ser Gly Lys Lys Ile Leu
1 5 10 15
Gly Phe Leu Phe Ile Val Cys Ser Leu Gly Leu Ser Ala Thr Val His
20 25 30
Gly Glu Thr Thr Asn Ser Gln Gln Leu Leu Ser Asn Asn Ile Asn Thr
35 40 45
Glu Leu Ile Asn His Asn Ser Asn Ala Ile Leu Ser Ser Thr Glu Gly
50 55 60
Ser Thr Thr Asp Ser Ile Asn Leu Gly Ala Gln Ser Pro Ala Val Lys
65 70 75 80
Ser Thr Thr Arg Thr Glu Leu Asp Val Thr Gly Ala Ala Lys Thr Leu
85 90 95
Leu Gln Thr Ser Ala Val Gln Lys Glu Met Lys Val Ser Leu Gln Glu
100 105 110
Thr Gln Val Ser Ser Glu Phe Ser Lys Arg Asp Ser Val Thr Asn Lys
115 120 125
Glu Ala Val Pro Val Ser Lys Asp Glu Leu Leu Glu Gln Ser Glu Val
130 135 140
Val Val Ser Thr Ser Ser Ile Gln Lys Asn Lys Ile Leu Asp Asn Lys
145 150 155 160
Lys Lys Arg Ala Asn Phe Val Thr Ser Ser Pro Leu Ile Lys Glu Lys
165 170 175
Pro Ser Asn Ser Lys Asp Ala Ser Gly Val Ile Asp Asn Ser Ala Ser
180 185 190
Pro Leu Ser Tyr Arg Lys Ala Lys Glu Val Val Ser Leu Arg Ile Glu
195 200 205
Gly Arg Gln Pro Leu Lys Asn Gln Lys Val Glu Ala Gln Pro Leu Leu
210 215 220
Ile Ser Asn Ser Ser Glu Lys Lys Ala Ser Val Tyr Thr Asn Ser His
225 230 235 240
Asp Phe Trp Asp Tyr Gln Trp Asp Met Lys Tyr Val Thr Asn Asn Gly
245 250 255
Glu Ser Tyr Ala Leu Tyr Gln Pro Ser Lys Lys Ile Ser Val Gly Ile
260 265 270
Ile Asp Ser Gly Ile Met Glu Glu His Pro Asp Leu Ser Asn Ser Leu
275 280 285
Gly Asn Tyr Phe Lys Asn Leu Val Pro Lys Gly Gly Phe Asp Asn Glu
290 295 300
Glu Pro Asp Glu Thr Gly Asn Pro Ser Asp Ile Val Asp Lys Met Gly
305 310 315 320
His Gly Thr Glu Val Ala Gly Gln Ile Thr Ala Asn Gly Asn Ile Leu
325 330 335
Gly Val Ala Pro Gly Ile Thr Val Asn Ile Tyr Arg Val Phe Gly Glu
340 345 350
Asn Leu Ser Lys Ser Glu Trp Val Ala Arg Ala Ile Arg Arg Ala Ala
355 360 365
Asp Asp Gly Asn Lys Val Ile Asn Ile Ser Ala Gly Gln Tyr Leu Met
370 375 380
Ile Ser Gly Ser Tyr Asp Asp Gly Thr Asn Asp Tyr Gln Glu Tyr Leu
385 390 395 400
Asn Tyr Lys Ser Ala Ile Asn Tyr Ala Thr Ala Lys Gly Ser Ile Val
405 410 415
Val Ala Ala Leu Gly Asn Asp Ser Leu Asn Ile Gln Asp Asn Gln Thr
420 425 430
Met Ile Asn Phe Leu Lys Arg Phe Arg Ser Ile Lys Val Pro Gly Lys
435 440 445
Val Val Asp Ala Pro Ser Val Phe Glu Asp Val Ile Ala Val Gly Gly
450 455 460
Ile Asp Gly Tyr Gly Asn Ile Ser Asp Phe Ser Asn Ile Gly Ala Asp
465 470 475 480
Ala Ile Tyr Ala Pro Ala Gly Thr Thr Ala Asn Phe Lys Lys Tyr Gly
485 490 495
Gln Asp Lys Phe Val Ser Gln Gly Tyr Tyr Leu Lys Asp Trp Leu Phe
500 505 510
Thr Thr Ala Asn Thr Gly Trp Tyr Gln Tyr Val Tyr Gly Asn Ser Phe
515 520 525
Ala Thr Pro Lys Val Ser Gly Ala Leu Ala Leu Val Val Asp Lys Tyr
530 535 540
Gly Ile Lys Asn Pro Asn Gln Leu Lys Arg Phe Leu Leu Met Asn Ser
545 550 555 560
Pro Glu Val Asn Gly Asn Arg Val Leu Asn Ile Val Asp Leu Leu Asn
565 570 575
Gly Lys Asn Lys Ala Phe Ser Leu Asp Thr Asp Lys Gly Gln Asp Asp
580 585 590
Ala Ile Asn His Lys Ser Met Glu Asn Leu Lys Glu Ser Arg Asp Thr
595 600 605
Met Lys Gln Glu Gln Asp Lys Glu Ile Gln Arg Asn Thr Asn Asn Asn
610 615 620
Phe Ser Ile Lys Asn Asp Phe His Asn Ile Ser Lys Glu Val Ile Ser
625 630 635 640
Val Asp Tyr Asn Ile Asn Gln Lys Met Ala Asn Asn Arg Asn Ser Arg
645 650 655
Gly Ala Val Ser Val Arg Ser Gln Glu Ile Leu Pro Val Thr Ile Glu
660 665 670
Gly Arg Gly Asp Gly Glu Asp Phe Leu Pro Ala Leu Gly Ile Val Cys
675 680 685
Ile Ser Ile Leu Gly Ile Leu Lys Arg Lys Thr Lys Asn His His His
690 695 700
His His His
705
<210> 4
<211> 729
<212> PRT
<213>artificial (Artificial)
<220>
<221>
<222>
<223>NisP substrate specificity proPAO
<400> 4
Met Val Gly Val Thr Val Ile Pro Arg Leu Leu Gly Leu Lys Asp Glu
1 5 10 15
Lys Ala Ser Pro Arg Ile Lys Ile Ala Thr Thr Val Gly Glu Ala Arg
20 25 30
Leu Ser Gly Ile Asn Tyr Arg His Pro Asp Ser Ala Leu Val Ser Tyr
35 40 45
Pro Val Ala Ala Ala Ala Pro Leu Gly Arg Leu Pro Ala Gly Asn Tyr
50 55 60
Arg Ile Ala Ile Val Gly Gly Gly Ala Gly Gly Ile Ala Ala Leu Tyr
65 70 75 80
Glu Leu Gly Arg Leu Ala Ala Thr Leu Pro Ala Gly Ser Gly Ile Asp
85 90 95
Val Gln Ile Tyr Glu Ala Asp Pro Asp Ser Phe Leu His Asp Arg Pro
100 105 110
Gly Ala Ser Pro Arg Ile Ala Ile Lys Val Arg Gly Leu Lys Ala Gly
115 120 125
Arg Val Ser Ala Ala Leu Val His Asn Gly Asp Pro Ala Ser Gly Asp
130 135 140
Thr Ile Tyr Glu Val Gly Ala Met Arg Phe Pro Glu Ile Ala Gly Leu
145 150 155 160
Thr Trp His Tyr Ala Ser Ala Ala Phe Gly Asp Ala Ala Pro Ile Lys
165 170 175
Val Phe Pro Asn Pro Gly Lys Val Pro Thr Glu Phe Val Phe Gly Asn
180 185 190
Arg Val Asp Arg Tyr Val Gly Ser Asp Pro Lys Asp Trp Glu Asp Pro
195 200 205
Asp Ser Pro Thr Leu Lys Val Leu Gly Val Val Ala Gly Gly Leu Val
210 215 220
Gly Asn Pro Gln Gly Glu Asn Val Ala Met Tyr Pro Ile Ala Asn Val
225 230 235 240
Asp Pro Ala Lys Ile Ala Ala Ile Leu Asn Ala Ala Thr Pro Pro Ala
245 250 255
Asp Ala Leu Glu Arg Ile Gln Thr Lys Tyr Trp Pro Glu Phe Ile Ala
260 265 270
Gln Tyr Asp Gly Leu Thr Leu Gly Ala Ala Val Arg Glu Ile Val Thr
275 280 285
Val Ala Phe Glu Lys Gly Thr Leu Pro Pro Val Asp Gly Val Leu Asp
290 295 300
Val Asp Glu Ser Ile Ser Tyr Tyr Val Glu Leu Phe Gly Arg Phe Gly
305 310 315 320
Phe Gly Thr Gly Gly Phe Lys Pro Leu Tyr Asn Ile Ser Leu Val Glu
325 330 335
Met Met Arg Leu Ile Leu Trp Asp Tyr Ser Asn Glu Tyr Thr Leu Pro
340 345 350
Val Thr Glu Asn Val Glu Phe Ile Arg Asn Leu Phe Leu Lys Ala Gln
355 360 365
Asn Val Gly Ala Gly Lys Leu Val Val Gln Val Arg Gln Glu Arg Val
370 375 380
Ala Asn Ala Cys His Ser Gly Thr Ala Ser Ala Arg Ala Gln Leu Leu
385 390 395 400
Ser Tyr Asp Ser His Asn Ala Val His Ser Glu Ala Tyr Asp Phe Val
405 410 415
Ile Leu Ala Val Pro His Asp Gln Leu Thr Pro Ile Val Ser Arg Ser
420 425 430
Gly Phe Glu His Ala Ala Ser Gln Asn Leu Gly Asp Ala Gly Leu Gly
435 440 445
Leu Glu Thr His Thr Tyr Asn Gln Val Tyr Pro Pro Leu Leu Leu Ser
450 455 460
Asp Ser Ser Pro Ala Ala Asn Ala Arg Ile Val Thr Ala Ile Gly Gln
465 470 475 480
Leu His Met Ala Arg Ser Ser Lys Val Phe Ala Thr Val Lys Thr Ala
485 490 495
Ala Leu Asp Gln Pro Trp Val Pro Gln Trp Arg Gly Glu Pro Ile Lys
500 505 510
Ala Val Val Ser Asp Ser Gly Leu Ala Ala Ser Tyr Val Val Pro Ser
515 520 525
Pro Ile Val Glu Asp Gly Gln Ala Pro Glu Tyr Ser Ser Leu Leu Ala
530 535 540
Ser Tyr Thr Trp Glu Asp Asp Ser Thr Arg Leu Arg His Asp Phe Gly
545 550 555 560
Leu Tyr Pro Gln Asn Pro Ala Thr Glu Thr Gly Thr Ala Asp Gly Met
565 570 575
Tyr Arg Thr Met Val Asn Arg Ala Tyr Arg Tyr Val Lys Tyr Ala Gly
580 585 590
Ala Ser Asn Ala Gln Pro Trp Trp Phe Tyr Gln Leu Leu Ala Glu Ala
595 600 605
Arg Thr Ala Asp Arg Phe Val Phe Asp Trp Thr Thr Asn Lys Thr Ala
610 615 620
Gly Gly Phe Lys Leu Asp Met Thr Gly Asp His His Gln Ser Asn Leu
625 630 635 640
Cys Phe Arg Tyr His Thr His Ala Leu Ala Ala Ser Leu Asp Asn Arg
645 650 655
Phe Phe Ile Ala Ser Asp Ser Tyr Ser His Leu Gly Gly Trp Leu Glu
660 665 670
Gly Ala Phe Met Ser Ala Leu Asn Ala Val Ala Gly Leu Ile Val Arg
675 680 685
Ala Asn Arg Gly Asp Val Ser Ala Leu Ser Thr Glu Ala Arg Pro Leu
690 695 700
Val Ile Gly Leu Arg Pro Val Val Lys Val Pro Ala Ala Glu Leu Ala
705 710 715 720
Thr Ser Gln His His His His His His
725
<210> 5
<211> 726
<212> PRT
<213>artificial (Artificial)
<220>
<221>
<222>
<223>Plasmin specificity proPAO
<400> 5
Met Val Gly Val Thr Val Ile Pro Arg Leu Leu Gly Leu Lys Asp Glu
1 5 10 15
Lys Gly Tyr Arg Ala Lys Ile Ala Thr Thr Val Gly Glu Ala Arg Leu
20 25 30
Ser Gly Ile Asn Tyr Arg His Pro Asp Ser Ala Leu Val Ser Tyr Pro
35 40 45
Val Ala Ala Ala Ala Pro Leu Gly Arg Leu Pro Ala Gly Asn Tyr Arg
50 55 60
Ile Ala Ile Val Gly Gly Gly Ala Gly Gly Ile Ala Ala Leu Tyr Glu
65 70 75 80
Leu Gly Arg Leu Ala Ala Thr Leu Pro Ala Gly Ser Gly Ile Asp Val
85 90 95
Gln Ile Tyr Glu Ala Asp Pro Asp Ser Phe Leu His Asp Arg Pro Gly
100 105 110
Gly Tyr Arg Ala Ile Lys Val Arg Gly Leu Lys Ala Gly Arg Val Ser
115 120 125
Ala Ala Leu Val His Asn Gly Asp Pro Ala Ser Gly Asp Thr Ile Tyr
130 135 140
Glu Val Gly Ala Met Arg Phe Pro Glu Ile Ala Gly Leu Thr Trp His
145 150 155 160
Tyr Ala Ser Ala Ala Phe Gly Asp Ala Ala Pro Ile Lys Val Phe Pro
165 170 175
Asn Pro Gly Lys Val Pro Thr Glu Phe Val Phe Gly Asn Arg Val Asp
180 185 190
Arg Tyr Val Gly Ser Asp Pro Lys Asp Trp Glu Asp Pro Asp Ser Pro
195 200 205
Thr Leu Lys Val Leu Gly Val Val Ala Gly Gly Leu Val Gly Asn Pro
210 215 220
Gln Gly Glu Asn Val Ala Met Tyr Pro Ile Ala Asn Val Asp Pro Ala
225 230 235 240
Lys Ile Ala Ala Ile Leu Asn Ala Ala Thr Pro Pro Ala Asp Ala Leu
245 250 255
Glu Arg Ile Gln Thr Lys Tyr Trp Pro Glu Phe Ile Ala Gln Tyr Asp
260 265 270
Gly Leu Thr Leu Gly Ala Ala Val Arg Glu Ile Val Thr Val Ala Phe
275 280 285
Glu Lys Gly Thr Leu Pro Pro Val Asp Gly Val Leu Asp Val Asp Glu
290 295 300
Ser Ile Ser Tyr Tyr Val Glu Leu Phe Gly Arg Phe Gly Phe Gly Thr
305 310 315 320
Gly Gly Phe Lys Pro Leu Tyr Asn Ile Ser Leu Val Glu Met Met Arg
325 330 335
Leu Ile Leu Trp Asp Tyr Ser Asn Glu Tyr Thr Leu Pro Val Thr Glu
340 345 350
Asn Val Glu Phe Ile Arg Asn Leu Phe Leu Lys Ala Gln Asn Val Gly
355 360 365
Ala Gly Lys Leu Val Val Gln Val Arg Gln Glu Arg Val Ala Asn Ala
370 375 380
Cys His Ser Gly Thr Ala Ser Ala Arg Ala Gln Leu Leu Ser Tyr Asp
385 390 395 400
Ser His Asn Ala Val His Ser Glu Ala Tyr Asp Phe Val Ile Leu Ala
405 410 415
Val Pro His Asp Gln Leu Thr Pro Ile Val Ser Arg Ser Gly Phe Glu
420 425 430
His Ala Ala Ser Gln Asn Leu Gly Asp Ala Gly Leu Gly Leu Glu Thr
435 440 445
His Thr Tyr Asn Gln Val Tyr Pro Pro Leu Leu Leu Ser Asp Ser Ser
450 455 460
Pro Ala Ala Asn Ala Arg Ile Val Thr Ala Ile Gly Gln Leu His Met
465 470 475 480
Ala Arg Ser Ser Lys Val Phe Ala Thr Val Lys Thr Ala Ala Leu Asp
485 490 495
Gln Pro Trp Val Pro Gln Trp Arg Gly Glu Pro Ile Lys Ala Val Val
500 505 510
Ser Asp Ser Gly Leu Ala Ala Ser Tyr Val Val Pro Ser Pro Ile Val
515 520 525
Glu Asp Gly Gln Ala Pro Glu Tyr Ser Ser Leu Leu Ala Ser Tyr Thr
530 535 540
Trp Glu Asp Asp Ser Thr Arg Leu Arg His Asp Phe Gly Leu Tyr Pro
545 550 555 560
Gln Asn Pro Ala Thr Glu Thr Gly Thr Ala Asp Gly Met Tyr Arg Thr
565 570 575
Met Val Asn Arg Ala Tyr Arg Tyr Val Lys Tyr Ala Gly Ala Ser Asn
580 585 590
Ala Gln Pro Trp Trp Phe Tyr Gln Leu Leu Ala Glu Ala Arg Thr Ala
595 600 605
Asp Arg Phe Val Phe Asp Trp Thr Thr Asn Lys Thr Ala Gly Gly Phe
610 615 620
Lys Leu Asp Met Thr Gly Asp His His Gln Ser Asn Leu Cys Phe Arg
625 630 635 640
Tyr His Thr His Ala Leu Ala Ala Ser Leu Asp Asn Arg Phe Phe Ile
645 650 655
Ala Ser Asp Ser Tyr Ser His Leu Gly Gly Trp Leu Glu Gly Ala Phe
660 665 670
Met Ser Ala Leu Asn Ala Val Ala Gly Leu Ile Val Arg Ala Asn Arg
675 680 685
Gly Asp Val Ser Ala Leu Ser Thr Glu Ala Arg Pro Leu Val Ile Gly
690 695 700
Leu Arg Pro Val Val Lys Val Pro Ala Ala Glu Leu Ala Thr Ser Gln
705 710 715 720
His His His His His His
725
<210> 6
<211> 727
<212> PRT
<213>artificial (Artificial)
<220>
<221>
<222>
<223>Caspase-3 substrate specificity proPAO
<400> 6
Met Val Gly Val Thr Val Ile Pro Arg Leu Leu Gly Leu Lys Asp Glu
1 5 10 15
Lys Asp Glu Val Asp Lys Ile Ala Thr Thr Val Gly Glu Ala Arg Leu
20 25 30
Ser Gly Ile Asn Tyr Arg His Pro Asp Ser Ala Leu Val Ser Tyr Pro
35 40 45
Val Ala Ala Ala Ala Pro Leu Gly Arg Leu Pro Ala Gly Asn Tyr Arg
50 55 60
Ile Ala Ile Val Gly Gly Gly Ala Gly Gly Ile Ala Ala Leu Tyr Glu
65 70 75 80
Leu Gly Arg Leu Ala Ala Thr Leu Pro Ala Gly Ser Gly Ile Asp Val
85 90 95
Gln Ile Tyr Glu Ala Asp Pro Asp Ser Phe Leu His Asp Arg Pro Gly
100 105 110
Asp Glu Val Asp Ala Ile Lys Val Arg Gly Leu Lys Ala Gly Arg Val
115 120 125
Ser Ala Ala Leu Val His Asn Gly Asp Pro Ala Ser Gly Asp Thr Ile
130 135 140
Tyr Glu Val Gly Ala Met Arg Phe Pro Glu Ile Ala Gly Leu Thr Trp
145 150 155 160
His Tyr Ala Ser Ala Ala Phe Gly Asp Ala Ala Pro Ile Lys Val Phe
165 170 175
Pro Asn Pro Gly Lys Val Pro Thr Glu Phe Val Phe Gly Asn Arg Val
180 185 190
Asp Arg Tyr Val Gly Ser Asp Pro Lys Asp Trp Glu Asp Pro Asp Ser
195 200 205
Pro Thr Leu Lys Val Leu Gly Val Val Ala Gly Gly Leu Val Gly Asn
210 215 220
Pro Gln Gly Glu Asn Val Ala Met Tyr Pro Ile Ala Asn Val Asp Pro
225 230 235 240
Ala Lys Ile Ala Ala Ile Leu Asn Ala Ala Thr Pro Pro Ala Asp Ala
245 250 255
Leu Glu Arg Ile Gln Thr Lys Tyr Trp Pro Glu Phe Ile Ala Gln Tyr
260 265 270
Asp Gly Leu Thr Leu Gly Ala Ala Val Arg Glu Ile Val Thr Val Ala
275 280 285
Phe Glu Lys Gly Thr Leu Pro Pro Val Asp Gly Val Leu Asp Val Asp
290 295 300
Glu Ser Ile Ser Tyr Tyr Val Glu Leu Phe Gly Arg Phe Gly Phe Gly
305 310 315 320
Thr Gly Gly Phe Lys Pro Leu Tyr Asn Ile Ser Leu Val Glu Met Met
325 330 335
Arg Leu Ile Leu Trp Asp Tyr Ser Asn Glu Tyr Thr Leu Pro Val Thr
340 345 350
Glu Asn Val Glu Phe Ile Arg Asn Leu Phe Leu Lys Ala Gln Asn Val
355 360 365
Gly Ala Gly Lys Leu Val Val Gln Val Arg Gln Glu Arg Val Ala Asn
370 375 380
Ala Cys His Ser Gly Thr Ala Ser Ala Arg Ala Gln Leu Leu Ser Tyr
385 390 395 400
Asp Ser His Asn Ala Val His Ser Glu Ala Tyr Asp Phe Val Ile Leu
405 410 415
Ala Val Pro His Asp Gln Leu Thr Pro Ile Val Ser Arg Ser Gly Phe
420 425 430
Glu His Ala Ala Ser Gln Asn Leu Gly Asp Ala Gly Leu Gly Leu Glu
435 440 445
Thr His Thr Tyr Asn Gln Val Tyr Pro Pro Leu Leu Leu Ser Asp Ser
450 455 460
Ser Pro Ala Ala Asn Ala Arg Ile Val Thr Ala Ile Gly Gln Leu His
465 470 475 480
Met Ala Arg Ser Ser Lys Val Phe Ala Thr Val Lys Thr Ala Ala Leu
485 490 495
Asp Gln Pro Trp Val Pro Gln Trp Arg Gly Glu Pro Ile Lys Ala Val
500 505 510
Val Ser Asp Ser Gly Leu Ala Ala Ser Tyr Val Val Pro Ser Pro Ile
515 520 525
Val Glu Asp Gly Gln Ala Pro Glu Tyr Ser Ser Leu Leu Ala Ser Tyr
530 535 540
Thr Trp Glu Asp Asp Ser Thr Arg Leu Arg His Asp Phe Gly Leu Tyr
545 550 555 560
Pro Gln Asn Pro Ala Thr Glu Thr Gly Thr Ala Asp Gly Met Tyr Arg
565 570 575
Thr Met Val Asn Arg Ala Tyr Arg Tyr Val Lys Tyr Ala Gly Ala Ser
580 585 590
Asn Ala Gln Pro Trp Trp Phe Tyr Gln Leu Leu Ala Glu Ala Arg Thr
595 600 605
Ala Asp Arg Phe Val Phe Asp Trp Thr Thr Asn Lys Thr Ala Gly Gly
610 615 620
Phe Lys Leu Asp Met Thr Gly Asp His His Gln Ser Asn Leu Cys Phe
625 630 635 640
Arg Tyr His Thr His Ala Leu Ala Ala Ser Leu Asp Asn Arg Phe Phe
645 650 655
Ile Ala Ser Asp Ser Tyr Ser His Leu Gly Gly Trp Leu Glu Gly Ala
660 665 670
Phe Met Ser Ala Leu Asn Ala Val Ala Gly Leu Ile Val Arg Ala Asn
675 680 685
Arg Gly Asp Val Ser Ala Leu Ser Thr Glu Ala Arg Pro Leu Val Ile
690 695 700
Gly Leu Arg Pro Val Val Lys Val Pro Ala Ala Glu Leu Ala Thr Ser
705 710 715 720
Gln His His His His His His
725
<210> 7
<211> 735
<212> PRT
<213>artificial (Artificial)
<220>
<221>
<222>
<223>C5 convertase substrate specificity proPAO
<400> 7
Met Val Gly Val Thr Val Ile Pro Arg Leu Leu Gly Leu Lys Asp Glu
1 5 10 15
Lys Gln Leu Gly Arg Leu His Met Lys Lys Ile Ala Thr Thr Val Gly
20 25 30
Glu Ala Arg Leu Ser Gly Ile Asn Tyr Arg His Pro Asp Ser Ala Leu
35 40 45
Val Ser Tyr Pro Val Ala Ala Ala Ala Pro Leu Gly Arg Leu Pro Ala
50 55 60
Gly Asn Tyr Arg Ile Ala Ile Val Gly Gly Gly Ala Gly Gly Ile Ala
65 70 75 80
Ala Leu Tyr Glu Leu Gly Arg Leu Ala Ala Thr Leu Pro Ala Gly Ser
85 90 95
Gly Ile Asp Val Gln Ile Tyr Glu Ala Asp Pro Asp Ser Phe Leu His
100 105 110
Asp Arg Pro Gly Gln Leu Gly Arg Leu His Met Lys Ala Ile Lys Val
115 120 125
Arg Gly Leu Lys Ala Gly Arg Val Ser Ala Ala Leu Val His Asn Gly
130 135 140
Asp Pro Ala Ser Gly Asp Thr Ile Tyr Glu Val Gly Ala Met Arg Phe
145 150 155 160
Pro Glu Ile Ala Gly Leu Thr Trp His Tyr Ala Ser Ala Ala Phe Gly
165 170 175
Asp Ala Ala Pro Ile Lys Val Phe Pro Asn Pro Gly Lys Val Pro Thr
180 185 190
Glu Phe Val Phe Gly Asn Arg Val Asp Arg Tyr Val Gly Ser Asp Pro
195 200 205
Lys Asp Trp Glu Asp Pro Asp Ser Pro Thr Leu Lys Val Leu Gly Val
210 215 220
Val Ala Gly Gly Leu Val Gly Asn Pro Gln Gly Glu Asn Val Ala Met
225 230 235 240
Tyr Pro Ile Ala Asn Val Asp Pro Ala Lys Ile Ala Ala Ile Leu Asn
245 250 255
Ala Ala Thr Pro Pro Ala Asp Ala Leu Glu Arg Ile Gln Thr Lys Tyr
260 265 270
Trp Pro Glu Phe Ile Ala Gln Tyr Asp Gly Leu Thr Leu Gly Ala Ala
275 280 285
Val Arg Glu Ile Val Thr Val Ala Phe Glu Lys Gly Thr Leu Pro Pro
290 295 300
Val Asp Gly Val Leu Asp Val Asp Glu Ser Ile Ser Tyr Tyr Val Glu
305 310 315 320
Leu Phe Gly Arg Phe Gly Phe Gly Thr Gly Gly Phe Lys Pro Leu Tyr
325 330 335
Asn Ile Ser Leu Val Glu Met Met Arg Leu Ile Leu Trp Asp Tyr Ser
340 345 350
Asn Glu Tyr Thr Leu Pro Val Thr Glu Asn Val Glu Phe Ile Arg Asn
355 360 365
Leu Phe Leu Lys Ala Gln Asn Val Gly Ala Gly Lys Leu Val Val Gln
370 375 380
Val Arg Gln Glu Arg Val Ala Asn Ala Cys His Ser Gly Thr Ala Ser
385 390 395 400
Ala Arg Ala Gln Leu Leu Ser Tyr Asp Ser His Asn Ala Val His Ser
405 410 415
Glu Ala Tyr Asp Phe Val Ile Leu Ala Val Pro His Asp Gln Leu Thr
420 425 430
Pro Ile Val Ser Arg Ser Gly Phe Glu His Ala Ala Ser Gln Asn Leu
435 440 445
Gly Asp Ala Gly Leu Gly Leu Glu Thr His Thr Tyr Asn Gln Val Tyr
450 455 460
Pro Pro Leu Leu Leu Ser Asp Ser Ser Pro Ala Ala Asn Ala Arg Ile
465 470 475 480
Val Thr Ala Ile Gly Gln Leu His Met Ala Arg Ser Ser Lys Val Phe
485 490 495
Ala Thr Val Lys Thr Ala Ala Leu Asp Gln Pro Trp Val Pro Gln Trp
500 505 510
Arg Gly Glu Pro Ile Lys Ala Val Val Ser Asp Ser Gly Leu Ala Ala
515 520 525
Ser Tyr Val Val Pro Ser Pro Ile Val Glu Asp Gly Gln Ala Pro Glu
530 535 540
Tyr Ser Ser Leu Leu Ala Ser Tyr Thr Trp Glu Asp Asp Ser Thr Arg
545 550 555 560
Leu Arg His Asp Phe Gly Leu Tyr Pro Gln Asn Pro Ala Thr Glu Thr
565 570 575
Gly Thr Ala Asp Gly Met Tyr Arg Thr Met Val Asn Arg Ala Tyr Arg
580 585 590
Tyr Val Lys Tyr Ala Gly Ala Ser Asn Ala Gln Pro Trp Trp Phe Tyr
595 600 605
Gln Leu Leu Ala Glu Ala Arg Thr Ala Asp Arg Phe Val Phe Asp Trp
610 615 620
Thr Thr Asn Lys Thr Ala Gly Gly Phe Lys Leu Asp Met Thr Gly Asp
625 630 635 640
His His Gln Ser Asn Leu Cys Phe Arg Tyr His Thr His Ala Leu Ala
645 650 655
Ala Ser Leu Asp Asn Arg Phe Phe Ile Ala Ser Asp Ser Tyr Ser His
660 665 670
Leu Gly Gly Trp Leu Glu Gly Ala Phe Met Ser Ala Leu Asn Ala Val
675 680 685
Ala Gly Leu Ile Val Arg Ala Asn Arg Gly Asp Val Ser Ala Leu Ser
690 695 700
Thr Glu Ala Arg Pro Leu Val Ile Gly Leu Arg Pro Val Val Lys Val
705 710 715 720
Pro Ala Ala Glu Leu Ala Thr Ser Gln His His His His His His
725 730 735
<210> 8
<211> 5
<212> PRT
<213>artificial (Artificial)
<220>
<221>
<222>
<223>the specific cutting sequence of prothrombin a
<220>
<221> MISC_FEATURE
<222> (1)..(2)
<223>" Xaa " is arbitrary amino acid residue
<220>
<221> MISC_FEATURE
<222> (3)..(3)
<223>" Xab " is the amino acid residue for preferably being selected from Pro, Ala, Gly or Val
<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223>" Xac " is the amino acid residue for preferably being selected from Ser, Ala, Gly etc.
<400> 8
Xaa Xaa Xab Arg Xac
1 5
<210> 9
<211> 4
<212> PRT
<213>artificial (Artificial)
<220>
<221>
<222>
<223>the specific cutting sequence 9 of prothrombin a
<400> 9
Leu Arg Pro Arg
1
<210> 10
<211> 5
<212> PRT
<213>artificial (Artificial)
<220>
<221>
<222>
<223>the specific cutting sequence 10 of prothrombin a
<400> 10
Leu Val Pro Arg Gly
1 5
<210> 11
<211> 3
<212> PRT
<213>artificial (Artificial)
<220>
<221>
<222>
<223>the specific cutting sequence 11 of prothrombin a
<400> 11
Phe Pro Arg
1
<210> 12
<211> 3
<212> PRT
<213>artificial (Artificial)
<220>
<221>
<222>
<223>the specific cutting sequence 12 of prothrombin a
<400> 12
Gly Arg Gly
1
<210> 13
<211> 4
<212> PRT
<213>artificial (Artificial)
<220>
<221>
<222>
<223>the specific cutting sequence of proconvertin a
<400> 13
Leu Ile Gln Arg
1
<210> 14
<211> 4
<212> PRT
<213>artificial (Artificial)
<220>
<221>
<222>
<223>the specific cutting sequence of factor IXa
<220>
<221> misc_feature
<222> (1)..(2)
<223>" Xaa " is arbitrary amino acid residue
<400> 14
Xaa Xaa Gly Arg
1
<210> 15
<211> 4
<212> PRT
<213>artificial (Artificial)
<220>
<221>
<222>
<223>the specific cutting sequence 15 of factor IXa
<400> 15
Pro Gln Gly Arg
1
<210> 16
<211> 4
<212> PRT
<213>artificial (Artificial)
<220>
<221>
<222>
<223>the specific cutting sequence of factor Xa
<220>
<221> MISC_FEATURE
<222> (1)..(1)
<223>" Xad " is the amino acid residue for preferably being selected from Ala or Ile
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223>" Xaa " is arbitrary amino acid residue
<220>
<221> MISC_FEATURE
<222> (3)..(3)
<223>" Xae " is the amino acid residue for preferably being selected from Pro, Phe or Gly
<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223>" Xaf " is the amino acid residue selected from Arg or Lys
<400> 16
Xad Xaa Xae Xaf
1
<210> 17
<211> 4
<212> PRT
<213>artificial (Artificial)
<220>
<221>
<222>
<223>the specific cutting sequence 17 of factor Xa
<400> 17
Ile Glu Gly Arg
1
<210> 18
<211> 4
<212> PRT
<213>artificial (Artificial)
<220>
<221>
<222>
<223>the specific cutting sequence 18 of factor Xa
<400> 18
Ile Asp Gly Arg
1
<210> 19
<211> 4
<212> PRT
<213>artificial (Artificial)
<220>
<221>
<222>
<223>the specific cutting sequence of plasma thromboplastin antecedent a
<220>
<221> MISC_FEATURE
<222> (1)..(1)
<223>" Xag " is the amino acid residue for preferably being selected from Lys or Asp
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223>" Xah " is the amino acid residue for preferably being selected from Phe or Leu
<400> 19
Xag Xah Thr Arg
1
<210> 20
<211> 4
<212> PRT
<213>artificial (Artificial)
<220>
<221>
<222>
<223>the specific cutting sequence 20 of plasma thromboplastin antecedent a
<400> 20
Lys Leu Thr Arg
1
<210> 21
<211> 4
<212> PRT
<213>artificial (Artificial)
<220>
<221>
<222>
<223>the specific cutting sequence of Hageman factor a
<400> 21
Thr Ser Thr Arg
1
<210> 22
<211> 4
<212> PRT
<213>artificial (Artificial)
<220>
<221>
<222>
<223>the specific cutting sequence of fibrinolysin Plasmin
<220>
<221> MISC_FEATURE
<222> (1)..(2)
<223>" Xaa " is arbitrary amino acid residue
<220>
<221> MISC_FEATURE
<222> (3)..(3)
<223>" Xai " is the amino acid residue for preferably being selected from Arg or Lys
<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223>" Xaj " is the amino acid residue for preferably being selected from Ala, Ser, Gly or Arg
<400> 22
Xaa Xaa Xai Xaj
1
<210> 23
<211> 4
<212> PRT
<213>artificial (Artificial)
<220>
<221>
<222>
<223>the specific cutting sequence 23 of fibrinolysin Plasmin
<400> 23
Gly Tyr Arg Ala
1
<210> 24
<211> 4
<212> PRT
<213>artificial (Artificial)
<220>
<221>
<222>
<223>the specific cutting sequence 24 of fibrinolysin Plasmin
<400> 24
Pro Ala Lys Ala
1
<210> 25
<211> 4
<212> PRT
<213>artificial (Artificial)
<220>
<221>
<222>
<223>the specific cutting sequence of Caspase-2
<220>
<221> MISC_FEATURE
<222> (3)..(3)
<223>" Xaa " is arbitrary amino acid residue
<400> 25
Asp Glu Xaa Asp
1
<210> 26
<211> 4
<212> PRT
<213>artificial (Artificial)
<220>
<221>
<222>
<223>the specific cutting sequence of Caspase-3
<220>
<221> MISC_FEATURE
<222> (1)..(1)
<223>" Xak " is the amino acid residue for preferably being selected from Asp or Glu
<400> 26
Xak Glu Val Asp
1
<210> 27
<211> 4
<212> PRT
<213>artificial (Artificial)
<220>
<221>
<222>
<223>the specific cutting sequence 27 of Caspase-3
<400> 27
Asp Glu Val Asp
1
<210> 28
<211> 4
<212> PRT
<213>artificial (Artificial)
<220>
<221>
<222>
<223>the specific cutting sequence of Caspase-6
<220>
<221> MISC_FEATURE
<222> (1)..(1)
<223>" Xaa " is arbitrary amino acid residue
<220>
<221> MISC_FEATURE
<222> (3)..(3)
<223>" Xaa " is arbitrary amino acid residue
<400> 28
Xaa Glu Xaa Asp
1
<210> 29
<211> 4
<212> PRT
<213>artificial (Artificial)
<220>
<221>
<222>
<223>the specific cutting sequence of Caspase-7
<220>
<221> MISC_FEATURE
<222> (2)..(3)
<223>" Xaa " is arbitrary amino acid residue
<400> 29
Asp Xaa Xaa Asp
1
<210> 30
<211> 4
<212> PRT
<213>artificial (Artificial)
<220>
<221>
<222>
<223>the specific cutting sequence of Caspase-8
<220>
<221> MISC_FEATURE
<222> (1)..(1)
<223>" Xal " is the amino acid residue for preferably being selected from Asp or Leu
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223>" Xam " is the amino acid residue for preferably being selected from Glu or Ser
<400> 30
Xal Xam Thr Asp
1
<210> 31
<211> 4
<212> PRT
<213>artificial (Artificial)
<220>
<221>
<222>
<223>the specific cutting sequence 31 of Caspase-8
<400> 31
Asp Glu Thr Asp
1
<210> 32
<211> 4
<212> PRT
<213>artificial (Artificial)
<220>
<221>
<222>
<223>the specific cutting sequence of Caspase-9
<220>
<221> MISC_FEATURE
<222> (1)..(1)
<223>" Xaa " is arbitrary amino acid residue
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223>" Xan " is the amino acid residue for preferably being selected from Asp or Glu
<220>
<221> MISC_FEATURE
<222> (3)..(3)
<223>" Xaa " is arbitrary amino acid residue
<400> 32
Xaa Xan Xaa Asp
1
<210> 33
<211> 4
<212> PRT
<213>artificial (Artificial)
<220>
<221>
<222>
<223>the specific cutting sequence of Caspase-10
<220>
<221> MISC_FEATURE
<222> (1)..(1)
<223>" Xaa " is arbitrary amino acid residue
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223>" Xao " is the amino acid residue for preferably being selected from Glu, Gln or Ser
<220>
<221> MISC_FEATURE
<222> (3)..(3)
<223>" Xap " is the amino acid residue for preferably being selected from Thr or Val
<400> 33
Xaa Xao Xap Asp
1
<210> 34
<211> 4
<212> PRT
<213>artificial (Artificial)
<220>
<221>
<222>
<223>the specific cutting sequence of Caspase-14
<220>
<221> MISC_FEATURE
<222> (3)..(3)
<223>" Xaa " is arbitrary amino acid residue
<400> 34
Leu Glu Xaa Asp
1
<210> 35
<211> 8
<212> PRT
<213>artificial (Artificial)
<220>
<221>
<222>
<223>the specific cutting sequence 35 of complement pathway C3/C5 invertase
<400> 35
Gln Leu Gly Arg Leu His Met Lys
1 5
<210> 36
<211> 8
<212> PRT
<213>artificial (Artificial)
<220>
<221>
<222>
<223>the specific cutting sequence 36 of complement pathway C3/C5 invertase
<400> 36
Gly Leu Ala Arg Ser Asn Leu Asp
1 5
<210> 37
<211> 4
<212> PRT
<213>artificial (Artificial)
<220>
<221>
<222>
<223>the specific cutting sequence of MMP-8
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223>" Xaq " is the amino acid residue for preferably being selected from Pro, Ala or Ser
<220>
<221> MISC_FEATURE
<222> (3)..(3)
<223>" Xaa " is arbitrary amino acid residue
<400> 37
Gly Xaq Xaa Gly
1
<210> 38
<211> 4
<212> PRT
<213>artificial (Artificial)
<220>
<221>
<222>
<223>the specific cutting sequence of MMP-11
<220>
<221> MISC_FEATURE
<222> (1)..(1)
<223>" Xaa " is arbitrary amino acid residue
<400> 38
Xaa Ala Ala Ala
1
<210> 39
<211> 4
<212> PRT
<213>artificial (Artificial)
<220>
<221>
<222>
<223>the specific cutting sequence of MMP-12
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223>" Xar " is the amino acid residue for preferably being selected from Pro, Ala or Gly
<220>
<221> MISC_FEATURE
<222> (3)..(4)
<223>" Xas " is the amino acid residue for preferably being selected from Ala or Gly
<400> 39
Gly Xar Xas Xas
1
<210> 40
<211> 5
<212> PRT
<213>artificial (Artificial)
<220>
<221>
<222>
<223>the specific cutting sequence of MMP-13
<220>
<221> MISC_FEATURE
<222> (3)..(3)
<223>" Xaa " is arbitrary amino acid residue
<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223>" Xat " is the amino acid residue for preferably being selected from Leu, Ile or Val
<400> 40
Gly Pro Xaa Gly Xat
1 5
<210> 41
<211> 5
<212> PRT
<213>artificial (Artificial)
<220>
<221>
<222>
<223>the specific cutting sequence 41 of MMP-13
<400> 41
Gly Pro Ala Gly Leu
1 5
<210> 42
<211> 5
<212> PRT
<213>artificial (Artificial)
<220>
<221>
<222>
<223>the specific cutting sequence of MMP-20
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223>" Xaa " is arbitrary amino acid residue
<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223>" Xau " is the amino acid residue for preferably being selected from Leu or Met
<400> 42
Pro Xaa Leu Pro Xau
1 5
<210> 43
<211> 5
<212> PRT
<213>artificial (Artificial)
<220>
<221>
<222>
<223>the specific cutting sequence 43 of MMP-20
<400> 43
Pro Ala Leu Pro Leu
1 5
<210> 44
<211> 5
<212> PRT
<213>artificial (Artificial)
<220>
<221>
<222>
<223>the specific cutting sequence 44 of MMP-20
<400> 44
Pro Ala Leu Pro Met
1 5
<210> 45
<211> 37
<212> DNA
<213>artificial (Artificial)
<220>
<221>
<222>
<223>upstream PAO A1-45
<400> 45
catgccatgg tgggcgttac cgtcattccc cggctgc 37
<210> 46
<211> 50
<212> DNA
<213>artificial (Artificial)
<220>
<221>
<222>
<223>downstream PAO A2-46
<400> 46
gcccgcgtac cttgatcgca cgtggacgca gtcccgggcg gtcatgaaga 50
<210> 47
<211> 50
<212> DNA
<213>artificial (Artificial)
<220>
<221>
<222>
<223>upstream PAO B1-47
<400> 47
tcttcatgac cgcccgggac tgcgtccacg tgcgatcaag gtacgcgggc 50
<210> 48
<211> 55
<212> DNA
<213>artificial (Artificial)
<220>
<221>
<222>
<223>downstream PAO B2-48
<400> 48
ggaattccat atgttaatga tgatgatgat gatgctggct ggtggccagc tccgc 55
<210> 49
<211> 50
<212> DNA
<213>artificial (Artificial)
<220>
<221>
<222>
<223>upstream PAO C1-49
<400> 49
cggcctgaag gacgagaagc tgcgtccacg taagattgcc accaccgttg 50
<210> 50
<211> 50
<212> DNA
<213>artificial (Artificial)
<220>
<221>
<222>
<223>downstream PAO C2-50
<400> 50
caacggtggt ggcaatctta cgtggacgca gcttctcgtc cttcaggccg 50
<210> 51
<211> 50
<212> DNA
<213>artificial (Artificial)
<220>
<221>
<222>
<223>downstream PAO A2-51
<400> 51
gcccgcgtac cttgatcgca atacgaggag atgctcccgg gcggtcatga 50
<210> 52
<211> 50
<212> DNA
<213>artificial (Artificial)
<220>
<221>
<222>
<223>upstream PAO B1-52
<400> 52
tcatgaccgc ccgggagcat ctcctcgtat tgcgatcaag gtacgcgggc 50
<210> 53
<211> 50
<212> DNA
<213>artificial (Artificial)
<220>
<221>
<222>
<223>upstream PAO C1-53
<400> 53
cctgaaggac gagaaggcat ctcctcgtat taagattgcc accaccgttg 50
<210> 54
<211> 50
<212> DNA
<213>artificial (Artificial)
<220>
<221>
<222>
<223>downstream PAO C2-54
<400> 54
caacggtggt ggcaatctta atacgaggag atgccttctc gtccttcagg 50
<210> 55
<211> 50
<212> DNA
<213>artificial (Artificial)
<220>
<221>
<222>
<223>downstream PAO A2-55
<400> 55
cagcccgcgt accttgatcg cacgataacc tcccgggcgg tcatgaagaa 50
<210> 56
<211> 50
<212> DNA
<213>artificial (Artificial)
<220>
<221>
<222>
<223>upstream PAO B1-56
<400> 56
ttcttcatga ccgcccggga ggttatcgtg cgatcaaggt acgcgggctg 50
<210> 57
<211> 50
<212> DNA
<213>artificial (Artificial)
<220>
<221>
<222>
<223>upstream PAO C1-57
<400> 57
cggcctgaag gacgagaagg gttatcgtgc aaagattgcc accaccgttg 50
<210> 58
<211> 50
<212> DNA
<213>artificial (Artificial)
<220>
<221>
<222>
<223>downstream PAO C2-58
<400> 58
caacggtggt ggcaatcttt gcacgataac ccttctcgtc cttcaggccg 50
<210> 59
<211> 50
<212> DNA
<213>artificial (Artificial)
<220>
<221>
<222>
<223>downstream PAO A2-59
<400> 59
gcccgcgtac cttgatcgca tcaacttcgt ctcccgggcg gtcatgaaga 50
<210> 60
<211> 50
<212> DNA
<213>artificial (Artificial)
<220>
<221>
<222>
<223>upstream PAO B1-60
<400> 60
tcttcatgac cgcccgggag acgaagttga tgcgatcaag gtacgcgggc 50
<210> 61
<211> 50
<212> DNA
<213>artificial (Artificial)
<220>
<221>
<222>
<223>upstream PAO C1-61
<400> 61
cggcctgaag gacgagaagg acgaagttga taagattgcc accaccgttg 50
<210> 62
<211> 50
<212> DNA
<213>artificial (Artificial)
<220>
<221>
<222>
<223>downstream PAO C2-62
<400> 62
caacggtggt ggcaatctta tcaacttcgt ccttctcgtc cttcaggccg 50
<210> 63
<211> 56
<212> DNA
<213>artificial (Artificial)
<220>
<221>
<222>
<223>downstream PAO A2-63
<400> 63
cgcgtacctt gatcgctttc atgtgcagac gacccagttg tcccgggcgg tcatga 56
<210> 64
<211> 56
<212> DNA
<213>artificial (Artificial)
<220>
<221>
<222>
<223>upstream PAO B1-64
<400> 64
tcatgaccgc ccgggacaac tgggtcgtct gcacatgaaa gcgatcaagg tacgcg 56
<210> 65
<211> 56
<212> DNA
<213>artificial (Artificial)
<220>
<221>
<222>
<223>upstream PAO C1-65
<400> 65
cctgaaggac gagaagcaac tgggtcgtct gcacatgaag attgccacca ccgttg 56
<210> 66
<211> 56
<212> DNA
<213>artificial (Artificial)
<220>
<221>
<222>
<223>downstream PAO C2-66
<400> 66
caacggtggt ggcaatcttc atgtgcagac gacccagttg cttctcgtcc ttcagg 56
<210> 67
<211> 5
<212> PRT
<213>artificial (Artificial)
<220>
<221>
<222>
<223>specific recognition sequence of NisP
<400> 67
Ala Ser Pro Arg Ile
1 5
<210> 68
<211> 4
<212> PRT
<213>artificial (Artificial)
<220>
<221>
<222>
<223>SplB proteolytic cleavage site
<400> 68
Trp Glu Leu Gln
1

Claims (13)

1. a kind of modified phenylalanine oxidizing ferment proenzyme, it is characterised in that: the modification is in phenylalanine oxidizing ferment enzyme The specific recognition site sequence of protease is introduced between leader sequence in former sequence and α subunit and between α subunit and β subunit.
2. modified phenylalanine oxidizing ferment proenzyme as described in claim 1, derives from Pseudomonas Sp.P- 501, East Lake pseudomonad (Pseudomonas donghuensis), Ralstonia solanacearum (Ralstonia Solanacearum) UW551 or onion Burkholderia (Burkholderia cepacia) ATCC 25416.
3. modified phenylalanine oxidizing ferment proenzyme as claimed in claim 1 or 2, the protease are selected from coagulation cascade egg White enzyme, such as prothrombin a, Va, VIIa, VIIIa, IXa, Xa, XIa or XIIa；Fibrinolysin；Asparagus fern ammonia containing cysteine Pepsin, for example, Caspase-1, Caspase-2, Caspase-3, Caspase-4, Caspase-5, Caspase-6, Caspase-7, Caspase-8, Caspase-9, Caspase-10 or Caspase-11；Complement pathway protein enzyme, such as the factor C1, C2, C3, C4, C5, C6, C7, C8, C9 or C3/C5 converting Enzyme；MMP family protein enzyme；NisP enzyme.
4. modified phenylalanine oxidizing ferment proenzyme as claimed in claim 1 or 2, the specific recognition sites of the protease Sequence such as SEQ ID NO:8-44,67 it is any shown in.
5. modified phenylalanine oxidizing ferment proenzyme according to any one of claims 1-4, the phenylalanine oxidizing ferment Proenzyme sequence is as shown in SEQ ID NO:1.
6. modified phenylalanine oxidizing ferment proenzyme as described in any one in claim 1-5, sequence such as SEQ ID NO: 2, shown in any one of 4-7.
7. modified phenylalanine oxidizing ferment proenzyme of any of claims 1-6 is for protease in test sample Active purposes.
8. purposes as claimed in claim 7, the sample is selected from body fluid, whole blood, blood plasma, serum or tissue.
9. a kind of isolated nucleic acid encodes modified phenylalanine oxidizing ferment described in any one of claim 1-6 Proenzyme.
10. a kind of expression vector, it includes isolated nucleic acid as claimed in claim 9.
11. the host cell for being converted or being transfected with expression vector described in any one of claim 10.
12. the kit of proteinase activity in a kind of test sample comprising described in any one of claims 1-6 modified Phenylalanine oxidizing ferment proenzyme.
13. kit as claimed in claim 12, the kit also includes modified NisP protease zymogens, preceding It leads between peptide and catalytic subunit comprising enzyme spcificity recognition site sequence, such as the specific recognition sequence of factor Xa.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020200315A1 (en) * 2019-04-03 2020-10-08 北京医院 Modified phenylalanine oxidase zymogen and use thereof
CN111909907A (en) * 2020-07-08 2020-11-10 浙江工业大学 Aspartate oxidase mutant, engineering bacteria and application of aspartate oxidase mutant in preparation of refined glufosinate-ammonium through oxidation-reduction coupling

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0458898A (en) * 1990-06-28 1992-02-25 Fujirebio Inc Tripeptide and activity measurement of angiotensin-conversion enzyme using same compound

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109929818B (en) * 2019-04-03 2021-03-16 北京医院 Modified phenylalanine oxidase zymogen and application thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0458898A (en) * 1990-06-28 1992-02-25 Fujirebio Inc Tripeptide and activity measurement of angiotensin-conversion enzyme using same compound

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020200315A1 (en) * 2019-04-03 2020-10-08 北京医院 Modified phenylalanine oxidase zymogen and use thereof
CN111909907A (en) * 2020-07-08 2020-11-10 浙江工业大学 Aspartate oxidase mutant, engineering bacteria and application of aspartate oxidase mutant in preparation of refined glufosinate-ammonium through oxidation-reduction coupling
CN111909907B (en) * 2020-07-08 2022-05-24 浙江工业大学 Aspartate oxidase mutant, engineering bacteria and application of aspartate oxidase mutant in preparation of refined glufosinate-ammonium through oxidation-reduction coupling

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